System and method for optogenetic therapy

ABSTRACT

Configurations are described for utilizing light-activated proteins within cell membranes and subcellular regions to assist with medical treatment paradigms, such as hypertension treatment via anatomically specific and temporally precise modulation of renal plexus activity. The invention provides for proteins, nucleic acids, vectors and methods for genetically targeted expression of light-sensitive proteins to specific cells or defined cell populations. In particular the invention provides systems, devices, and methods for millisecond-timescale temporal control of certain cell activities using moderate light intensities, such as the generation or inhibition of electrical spikes in nerve cells and other excitable cells.

RELATED APPLICATION DATA

This is a continuation application of U.S. patent application Ser. No.15/803,473, filed on Nov. 3, 2017, which is a continuation of U.S.patent application Ser. No. 15/473,458, filed on Mar. 29, 2017, which isa continuation of U.S. patent application Ser. No. 14/449,081, filed onJul. 31, 2014, which is a continuation application of InternationalApplication No. PCT/US2013/000262, filed on Nov. 21, 2013, which claimspriority to U.S. Provisional Application Ser. No. 61/729,283, filed onNov. 21, 2012. The foregoing applications are hereby incorporated byreference into the present application in their entirety. Priority tothe aforementioned applications is hereby expressly claimed inaccordance with 35 U.S.C. § § 119, 120, and 365 and any other applicablestatutes.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewith,and identified as follows: One 122 KiloByte ASCII (Text) file named“14_449_081_SeqList_ST25.txt” created on Sep. 18, 2018.

FIELD OF THE INVENTION

The present invention relates generally to systems, devices, andprocesses for facilitating various levels of control over cells andtissues in vivo, and more particularly to systems and methods forphysiologic intervention wherein light may be utilized as an input totissues which have been modified to become light sensitive.

BACKGROUND

Pharmacological and direct electrical neuromodulation techniques havebeen employed in various interventional settings to address challengessuch as prolonged orthopaedic pain, epilepsy, and hypertension.Pharmacological manipulations of the neural system may be targeted tocertain specific cell types, and may have relatively significantphysiologic impacts, but they typically act on a time scale of minutes,whereas neurons physiologically act on a time scale of milliseconds.Electrical stimulation techniques, on the other hand, may be moreprecise from an interventional time scale perspective, but theygenerally are not cell type specific and may therefore involvesignificant clinical downsides. A new neurointerventional field termed“Cytogenetics” is being developed which involves the use oflight-sensitive proteins, configurations for delivering related genes ina very specific way to targeted cells, and targeted illuminationtechniques to produce interventional tools with both low latency from atime scale perspective, and also high specificity from a cell typeperspective.

For example, optogenetic technologies and techniques recently have beenutilized in laboratory settings to change the membrane voltagepotentials of excitable cells, such as neurons, and to study thebehavior of such neurons before and after-exposure to light of variouswavelengths. In neurons, membrane depolarization leads to the activationof transient electrical signals (also called action potentials or“spikes”), which are the basis of neuronal communication. Conversely,membrane hyperpolarization leads to the inhibition of such signals. Byexogenously expressing light-activated proteins that change the membranepotential in neurons, light can be utilized as a triggering means toinduce inhibition or excitation.

One approach is to utilize naturally-occurring genes that encodelight-sensitive proteins, such as the so-called “opsins”. Theselight-sensitive transmembrane proteins may be covalently bonded tochromophore retinal, which upon absorption of light, isomerizes toactivate the protein. Notably, retinal compounds are found in mostvertebrate cells in sufficient quantities, thus eliminating the need toadminister exogenous molecules for this purpose. The first geneticallyencoded system for optical control in mammalian neurons usinglight-sensitive signaling proteins was established in Drosophilamelanogaster, a fruit fly species, and neurons expressing such proteinswere shown to respond to light exposure with waves of depolarization andspiking. More recently it has been discovered that opsins frommicroorganisms which combine the light-sensitive domain with an ion pumpor ion channel in the same protein may also modulate neuronal signalingto facilitate faster control in a single, easily-expressed, protein. In2002, it was discovered that a protein that causes green algae(Chlamydomonas reinhardtii) to move toward areas of light exposure is alight-sensitive channel; exposure to light of a particular wavelength(maximum results at blue light spectrum i.e., about 480 nm) for theopsin ChR2, also known as “channelrhodopsin”) causes the membranechannel to open, allowing positive ions, such as sodium ions, to floodinto the cell, much like the influx of ions that cause nerve cells tofire. Various other excitatory opsins, such as Volvox Channelrhodopsin(“VChR1”), Step Function Opsins (or “SFO”; ChR2 variants which canproduce prolonged, stable, excitable states with blue-wavelength lightexposure, and be reversed with exposure to green-wavelength light, i.e.,about 590 nm), or red-shifted optical excitation variants, such as“C1V1”, have been described by Karl Deisseroth and others, such as atthe opsin sequence information site hosted at the URL:http://www.stanford.edu/group/dlab/optogenetics/sequence_info.html, thecontent of which is incorporated by reference herein in its entirety.Examples of opsins are described in U.S. patent application Ser. Nos.11/459,638, 12/988,567, 12/522,520, and 13/577,565, and in Yizhar et al.2011, Neuron 71:9-34 and Zhang et al. 2011, Cell 147:1446-1457, all ofwhich are incorporated by reference herein in their entirety.

While excitation is desirable in some clinical scenarios, such as toprovide a perception of a sensory nerve stimulation equivalent,relatively high-levels of excitation may also be utilized to provide thefunctional equivalent of inhibition in an “overdrive” or“hyperstimulation” configuration. For example, a hyperstimulationconfiguration has been utilized with capsaicin, the active component ofchili peppers, to essentially overdrive associated pain receptors in amanner that prevents pain receptors from, otherwise delivering painsignals to the brain (i.e., in an analgesic indication). An example ofclinical use of hyperstimulation is the Brindley anterior sacral nerveroot stimulator for electrical stimulation of bladder emptying (Brindleyet al. Paraplegia 1982 20:365-381; Brindley et al. Journal of Neurology,Neurosurgery, and Psychiatry 1986 49:1104-1114; Brindley Paraplegia 199432:795-805; van der Aa et al. Archives of Physiology and Biochemistry1999 107:248-256; Nosseir et al. Neurourology and Urodynamics 200726:228-233; Martens et al. Neurourology and Urodynamics 201130:551-555). In a parallel manner, hyperstimulation or overdriving ofexcitation with an excitatory opsin configuration may provide inhibitoryfunctionality. It may also be referred to as a hyperstimulation blockwhen used to produce a depolarization block.

Other opsin configurations have been found to directly inhibit signaltransmission without hyperstimulation or overdriving. For example, lightstimulation of halorhodopsin (“NpHR”), a chloride ion pump,hyperpolarizes neurons and directly inhibits spikes in response toyellow-wavelength (˜589 nm) light irradiation. Other more recentvariants (such as those termed “eNpHR2.0” and “eNpHR3.0”) exhibitimproved membrane targeting and photocurrents in mammalian cells. Lightdriven proton pumps such as archaerhodopsin-3 (“Arch”) and “eARCH”, andArchT, Leptosphaeria maculans fungal opsins (“Mac”), enhancedbacteriorhodopsin (“eBR”), and Guillardia theta rhodopsin-3 (“GtR3”) mayalso be utilized to hyperpolarize neurons and block signaling. Directhyperpolarization is a specific and physiological intervention thatmimics normal neuronal inhibition. Suitable inhibitory opsins are alsodescribed in the aforementioned incorporated by reference resources.

Further, a ChR2 variant known as a Stabilized Step Function Opsin (or“SSFO”) provides light-activated ion channel functionality that caninhibit neural activity by depolarization block at the level of theaxon. This occurs when the depolarization results in a depolarizedmembrane potential such that sodium channels are inactivated and noaction potential of spikes can be generated.

C1V1-T refers to C1V1 (E122T) or C1V1 (E162T). C1V1-TT refers to C1V1(E122T/E162T).

The term light-sensitive protein, as used herein, refers to all theaforementioned types of ion channels and ion transporters/pumps in thecontext of modulating a membrane potential.

With a variety of opsins available for optogenetic experimentation inthe laboratory, there is a need to bring such technologies to the stageof medical intervention, which requires not only a suitable selection ofopsin-based tools for excitation and/or inhibition, but also a means fordelivering the genetic material to the subject patient and a means forcontrollably illuminating the subject tissue within the patient toutilize the light-driven capabilities. There is a need for practicalconfigurations and techniques for utilizing optogenetic technologies inthe clinical setting to address various clinical challenges of modernmedicine with specificity and temporal control precision.

SUMMARY OF THE INVENTION

One embodiment is directed to a method for stimulating a tissuestructure comprising light sensitive protein, which may compriseselecting a targeted tissue structure to be stimulated usingillumination at a first subcutaneous location, providing an implantablelight conductor configured to be permanently coupled between the firstsubcutaneous location and a second location selected such thatextracorporeal photons directed toward the second location may betransmitted, at least in part, through the implantable light conductorto the targeted tissue structure. Selecting the targeted tissuestructure to be stimulated may comprise imaging the target tissue usinga modality selected from the group consisting of: radiography,fluoroscopy, ultrasound, magnetic resonance imaging, computed tomographyimaging, and endoscopy. The method further may comprise providing anextracorporeal light source to direct photons through the implantablelight conductor to the targeted tissue structure. The implantable lightconductor may have a proximal end at the second location comprising anenlarged light collection surface. The enlarged light collection surfacemay comprise a wedge-shaped geometry with an entrance facet oriented tocapture the extracorporeal photons. The implantable light conductor maycomprise a waveguide configured to propagate substantially all lightthat is passed through it via total internal reflection. The implantablelight conductor may comprise a material type selected from the groupconsisting of: glasses, polymers, crystals. The implantable lightconductor may comprise a polymer selected from the group consisting of:poly methyl methacrylate, silicone, polydimethylsiloxane, and copolymersthereof. The implantable light conductor may comprise a reflective layerconfigured to recycle light that escapes total internal reflection as itis being propagated down the implantable light conductor. The reflectivelayer may comprise material selected from the group consisting ofsilver, rhodium, aluminum, and gold. The implantable light conductor maybe at least partially encapsulated with an insulating layer to protectthe implantable light conductor or other layers thereupon from theenvironment. The insulating layer may comprise a material selected fromthe group consisting of: silicon dioxide, aluminum oxide, and magnesiumdifluoride. The implantable light conductor may comprise a claddinglayer configured to confine evanescent waves within the implantablelight conductor as photons are propagated down the implantable lightconductor. The cladding layer may comprise material selected from thegroup consisting of: fluorinated ethylene propylene, polymethylpentene,and THV fluoropolymer blend. The implantable light conductor maycomprise a bioinert layer configured to improve biocompatibility andprevent changes to the refractive properties of the implantable lightconductor. The bioinert layer may comprise material selected from thegroup consisting of: gold, platinum, parylene-C, poly(ethylene glycol),phosphoryl choline, polyethylene oxide polymer, andD-mannitol-terminated alkanethiol. The method further may compriseinstalling a pilot member before insertion of the implantable lightconductor, the pilot member configured to assist in preparing theassociated tissue for implantation of the implantable light conductor.The pilot member may comprise a cutting tool or dilator. The methodfurther may comprise providing a delivery conduit defining a lumentherethrough through which the implantable light conductor may beremovably coupled. The method further may comprise providing animplantable light applicator configured to be coupled to the tissuestructure, and also coupled to at least one surface of the implantablelight conductor at the first location such that photons travellingthrough the implantable light conductor may be transferred into theimplantable light applicator to be directed into the tissue structure.The second location may be entirely encapsulated by tissue, and whereinthe implantable light conductor is configured to receive the photonsfrom the extracorporeal light source through a relatively thin layer oftissue. The relatively thin layer of tissue may have a maximum thicknessof between about 100 microns and about 1 millimeter. The second locationmay be directly extracorporeally accessible. The tissue structurecomprising the light sensitive protein may be genetically modified toencode an opsin protein. The opsin protein may be an inhibitory opsinprotein. The inhibitory opsin protein may be selected from the groupconsisting of: NpHR, eNpHR 1.0, eNpHR 2.0, eNpHR 3.0, Mac, Mac 3.0,Arch, and ArchT. The opsin protein may be a stimulatory opsin protein.The stimulatory opsin protein may be selected from the group consistingof: ChR2, C1V1-T, C1V1-TT, CatCh, VChR1-SFO, and ChR2-SFO.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an embodiment of a technique for optogenetic treatment ofa human in accordance with the present invention.

FIGS. 2A and 2B depict an embodiment of an injection configuration foroptogenetic treatment of a human in accordance with the presentinvention.

FIG. 3 depicts an embodiment of a system level componentry configurationfor optogenetic treatment of a human in accordance with the presentinvention.

FIGS. 4A and 4B depict activation wavelength and timing charts forvarious opsin proteins that may be utilized in embodiments of thepresent invention.

FIG. 4C (FIGS. 4C-1 and 4C-2) depicts an LED specification table forvarious LEDs that may be utilized in embodiments of the presentinvention.

FIG. 5 depicts an embodiment of one portion of an illuminationconfiguration for optogenetic treatment of a human in accordance withthe present invention.

FIG. 6 depicts a light power density chart that may be applied inembodiments of the present invention.

FIG. 7 depicts an irradiance versus geometry chart that may be appliedin embodiments of the present invention.

FIGS. 8-28 depict various aspects of embodiments of light deliveryconfigurations which may be utilized for optogenetic treatment of ahuman in accordance with the present invention.

FIGS. 29A and 29B illustrate system level deployments of optogenetictreatment systems for nerve root intervention in accordance with thepresent invention.

FIGS. 30A-39C depict various aspects of embodiments of light deliveryconfigurations and related issues and data, which may be utilized foroptogenetic treatment of a human in accordance with the presentinvention.

FIGS. 40A-50 depict various aspects of embodiments of configurationswhich may be utilized for optogenetic treatment of a hypertension usingneuromodulation of the renal plexus in accordance with the presentinvention.

FIGS. 51A-61M-7 depict various amino acid sequences of exemplary opsins,signal peptides, signal sequences, ER export sequences, and atrafficking sequence, as well as a polynucleotide sequence encodingChamp.

FIGS. 62A-62J-2 depict tables and charts containing descriptions of atleast some of the opsins described herein.

FIG. 63 depicts an embodiment of light delivery configurations for usewhen treating the renal pelvis connectors in accordance with the presentinvention.

FIGS. 64-68 depict various aspects of embodiments of optical and/orelectronic connectors in accordance with the present invention.

FIG. 69 depicts an embodiment of optical sensing in accordance with thepresent invention.

FIG. 70 depicts an embodiment of a percutaneous feedthrough inaccordance with the present invention.

FIGS. 71A-72B depict various aspects of embodiments of configurationswhich may be utilized for optogenetic treatment of the spine inaccordance with the present invention.

FIGS. 73A-75 depict various aspects of embodiments of configurations ofoptical feedthroughs in accordance with the present invention.

FIGS. 76A-78 depict various aspects of embodiments of configurationswhich may be utilized for optogenetic treatment of the spine inaccordance with the present invention.

FIGS. 79-81 depict various aspects of embodiments of light deliveryconfigurations and related issues and data, which may be utilized foroptogenetic treatment of a human in accordance with the presentinvention.

FIGS. 82A-83 depict various aspects of embodiments of light deliverystrain relief configurations and related issues and data, which may beutilized for optogenetic treatment of a human in accordance with thepresent invention.

FIGS. 84-86 depict various aspects of embodiments of in-vivo lightcollection configurations and related issues and data, which may beutilized for optogenetic treatment of a human in accordance with thepresent invention.

FIG. 87 depicts an embodiment for mounting an external charging deviceof a percutaneous feedthrough in accordance with the present invention.

FIGS. 88A-89 depict embodiments of an elongate member for use in thesurgical implantation of optogenetic therapeutic devices in accordancewith the present invention.

FIGS. 90-101 depict various configurations for conducting proceduresfeaturing technologies such as those described herein.

FIG. 102 depicts an embodiment of a system in accordance with thepresent invention.

DETAILED DESCRIPTION

Referring to FIG. 1, from a high-level perspective, anoptogenetics-based neuromodulation intervention involves determinationof a desired nervous system functional modulation which can befacilitated by optogenetic excitation and/or inhibition (2), followed bya selection of neuroanatomic resource within the patient to provide suchoutcome (4), delivery of an effective amount of polynucleotidecomprising a light-responsive opsin protein which is expressed inneurons of the targeted neuroanatomy (6), waiting for a period of timeto ensure that sufficient portions of the targeted neuroanatomy willindeed express the light responsive opsin protein upon exposure to light(8), and delivering light to the targeted neuroanatomy to causecontrolled, specific excitation and/or inhibition of such neuroanatomyby virtue of the presence of the light-responsive opsin protein therein(10).

While the development and use of transgenic animals has been utilized toaddress some of the aforementioned challenges, such techniques are notsuitable in human medicine. Means to deliver the light-responsive opsinto cells in vivo are required; there are a number of potentialmethodologies that can be used to achieve this goal. These include viralmediated gene delivery, electroporation, ultrasound, hydrodynamicdelivery, or the introduction of naked DNA. either by direct injectionor complemented by additional facilitators such as cationic lipids orpolymers. Minicircle DNA. technology may also be used. Minicircles areepisomal DNA vectors that may be produced as circular expressioncassettes devoid of any bacterial plasmid DNA backbone. Their smallermolecular size may enable more efficient transfections and offersustained expression.

Viral expression techniques, generally comprising delivery of DNAencoding a desired opsin and promoter/catalyst sequence packaged withina recombinant viral vector have been utilized with success in mammals toeffectively transfect targeted neuroanatomy and deliver genetic materialto the nuclei of targeted neurons, thereby inducing such neurons toproduce light-sensitive proteins which are migrated throughout theneuron cell membranes where they are made functionally available toillumination components of the interventional system. Typically a viralvector will package what may be referred to as an “opsin expressioncassette”, which will contain the opsin (e.g., ChR2, NpHR, etc.) and apromoter that will be selected to drive expression of the particularopsin. In the case of Adeno-associated virus (or AAV), the gene ofinterest (opsin) can be in a single stranded configuration with only oneopsin expression cassette or in a self-complementary structure with twocopies of opsin expression cassette complimentary in sequence with oneanother and connected by hairpin loops. The self-complementary AAVs arethought to be more stable and show higher expression levels. Thepromoter may confer specificity to a targeted tissue, such as in thecase of the human synapsin promoter (“hSyn”) or the human Thy1 promoter(“hThy1”) which allow protein expression of the gene under its controlin neurons. Another example is the calcium/calmodulin-dependent kinaseII promoter (“CAMKII”), which allows protein expression of the geneunder its control only in excitatory neurons, a subset of the neuronpopulation. Alternatively, a ubiquitous promoter may be utilized, suchas the human cytomegalovirus (“CMV”) promoter, or the chicken beta-actin(“CBA”) promoter, each of which is not particularly neural specific, andeach of which has been utilized safely in gene therapy trials forneurodegenerative disease. Alternatively, a combination of chickenbeta-actin promoter and cytomegalovirus immediate-early enhancer, knownas CAG promoter, may be utilized. Alternatively, a promoter domainderived from transcription factor Hb9, “survival of motor neuron”(SMN1), and methyl-CpG-binding protein-2 (MeCP2) may be utilized.Alternatively, MCK (muscle creatine kinase) promoter, MCK/SV40 promoter,Troponin promoter, and promoters of the transcription factors Pax6,Nkx6.1, Olig2, and Mnr2 may be utilized. Alternatively, a promoter suchas latency-associated promoter 2 (LAP2) or neuron-specific enolase (NSE)may be utilized. Alternatively, a human elongation factor-1 alpha EF1αpromoter may be utilized, including for example those from isoformsEF1α₁ and EF1α₂. EF1α₁ promoter may confer expression in brain,placenta, lung, liver, kidney, and pancreas. EF1α₂ promoter may conferexpression in terminally differentiated cells of the brain, heart, andskeletal muscle. In another embodiment, β-cell-specific rat insulinpromoter (RIP) may be utilized. In another embodiment, amacrophage-specific transcription promoter (such as CD68, or a truncatedversion thereof) may be utilized. Alternatively, a promoter such ashGFAP (for example, to direct expression to astrocytes), TPH-2 (forexample, to direct expression to Raphe serotonergic neurons), fugu SSTpromoter (fSST) (for example to direct expression to inhibitoryneurons), MBP (for example to direct expression to oligodendrocytes), ormouse SST (to direct expression to preBotzinger C somatostatin neurons),may be utilized. Viral constructs carrying opsins are optimized forspecific neuronal populations and are not limited to such illustrativeexamples.

Viral expression systems have the dual advantages of fast and versatileimplementation combined with high infective/copy number for robustexpression levels in targeted neuroanatomy. Cellular specificity may beobtained with viruses by virtue of promoter selection if the promotersare small, specific, and strong enough, by localized targeting of virusinjection, as discussed in further detail below, and by restriction ofopsin activation (i.e., via targeted illumination) of particular cellsor projections of cells, also as described in further detail below. Inan embodiment, an opsin is targeted by methods described in Yizhar etal. 2011, Neuron 71:9-34. In addition, different serotypes of the virus(conferred by the viral capsid or coat proteins) will show differenttissue trophism, Lenti- and adeno-associated (“AAV”) viral vectors havebeen utilized successfully to introduce opsins into the mouse, rate andprimate brain. Additionally, these have been well tolerated and highlyexpressed over relatively long periods of time with no reported adverseeffects, providing the opportunity for long-term treatment paradigms.Lentivirus, for example, is easily produced using standard tissueculture and ultracentrifuge techniques, while AAV may be reliablyproduced either by individual laboratories or through core viralfacilities. Viruses have been utilized to target many tissue structuresand systems, including but not limited to hypocretin neurons in thehypothalamus, excitatory pyramidal neurons, basal ganglia dopaminergicneurons, striatal GABAergic neurons, amygdala glutamatergic neurons,prefrontal cortical excitatory neurons and others, as well as astroglia.For example, it has been shown that the use of AAV-delivered ChR2 tocontrol astroglial activity in the brainstem of mice and create amechanism by which astroglia can transfer systemic information from theblood to neurons underlying homeostasis, in this case directlymodulating neurons that manipulate the rate of respiration. AAV is apreferred vector due to its safety profile, and AAV serotypes 1 and 6have been shown to infect motor neurons following intramuscularinjection in primates. Other vectors include but are not limited toequine infectious anemia virus pseudotyped with a retrograde transportprotein (e.g., Rabies G protein), and herpes simplex virus (“HSV”).

Referring back to FIG. 1, delivery of the polynucleotide comprising thelight-responsive opsin protein to be expressed in neurons of thetargeted neuroanatomy may involve injection with a syringe or otherdevice, in one or more configurations, including but not limited tointramuscular injection (i.e., straight into the muscle belly associatedwith a targeted portion of neuroanatomy), intraparenchymal injection(i.e., injection into the parenchyma of an organ such as the kidneywhich may be associated with a targeted portion of neuroanatomy), tissuestructure injection (i.e., injection into the wall of the stomach in thevicinity of a targeted portion of neuroanatomy such as stretch receptorsof the gastric nerve), intrafascicular injection (i.e., injectiondirectly into a targeted nerve or bundle thereof, such as into the vagusnerve), nerve ganglion injection (i.e., injection directly into theganglion, which comprises nerve cell bodies), vascular wall injection(i.e., injection of a vascular structure such as a renal artery wall inthe vicinity of a targeted portion of neuroanatomy such as the renalnerve plexus), and internal topical injection or application (i.e.,injection upon a surface of a tissue structure associated with atargeted portion of neuroanatomy, or upon the neuroanatomy itself,generally after surgical access, such as via laparoscopic techniques),Each of these injection configurations is explored in further detailbelow.

Intramuscular delivery is a common technique for delivering genes toperipheral neurons. The muscle may be palpitated by the surgeon oroperator to identify location and shape of the muscle. This informationmay then used to discern the likely site of the nerve endings (usinganatomical knowledge). A hypodermic needle (e.g., 23G to 27G) may beinserted transcutaneously into the muscle tissue of the pertinent musclein the vicinity of the nerve endings, and the vector solution may beinjected through the needle where it may diffuse throughout the muscletissue and be taken up by the nerve terminals (i.e. motor or sensoryneurons). The vector solution may be injected as a single bolus dose, orslowly through an infusion pump (e.g., at a rate of between about 0.01and 1.00 mL/min). An ultrasound guidance system may be used for deepermuscle targets. Once taken up by the neural terminals, the vectorpreferably is retrogradely transported to the pertinent neural cellbodies across the length of the axon. The number of injections and doseof virus injected to each muscle is dependent upon the muscle volume andtopology. In the non-human primate study described in Towne et al GeneTher. 2010 January; 17(1):141-6, a single injection of 1 mL salinesolution containing 1.3×10¹² viral genomes (vg) of AAV6 was injectedinto the triceps surae muscle (approximately 30 cm³) to achieveefficient transduction of the entire motor neuron pool.

In one example of an intramuscular injection therapy step for targetingmotor neurons for addressing spasticity problems, the flexor carpiulnaris muscle (having a volume of approximately 40 cubic centimeters)of the hand may be injected intramuscularly. This muscle may be targetedwith one to two injections containing a total of about 1 mL salinesolution containing 10¹² to 10¹³ vg. Larger muscles, such as the bicepsbrachii which has an approximate volume of 150 cubic centimeters, mayrequire two to five injections with a higher total dose of vector 5×10¹²to 10¹⁴ vg in 1 to 5 mL. These ranges are illustrative, and doses aretested for each virus-promoter-opsin construct pairing them with thetargeted neurons.

In another example of an intramuscular injection therapy step fortargeting motor neurons for addressing urinary system problems, theexternal urethral sphincter (“EUS”) may be injected intramuscularly withmultiple injections around the circumference of the tissue structure(which has a volume between 1 and about 5 cubic centimeters in the adulthuman). For example, in one embodiment, this tissue structure may beinjected using 4 or 5 injections with a total dose of vector 10¹² to10¹³ vg in 0.1 to 15 mL in rodents at the rate of 1 ml/min. For largeranimals and humans, larger volumes and titers may be used that would beempirically determined.

Vector delivery into the parenchyma of a tissue has been shown tofacilitate the targeting of neurons innervating that structure. A needlemay be inserted into the parenchyma of a pertinent organ (e.g., akidney) in the vicinity of the neural nerve endings (e.g., the renalnerve plexus). The vector solution may be injected through the needlewhere it may diffuse throughout the tissue and be taken up by the neuralterminals (i.e., sympathetic or parasympathetic nerve endings). Oncetaken up by the neural terminals, the vector may be retrogradelytransported to the neural cell body along the length of the axon. In oneembodiment, intraparenchymal injections are performed throughlaparoscopic surgical access and instrumentation. A small incision maybe made through the skin and other pertinent tissue structures (such asthe abdominal wall) to allow insertion of the surgical apparatus(camera, needle, tools, etc.). The needle may be guided into theparenchyma of the organ (as visualized through the camera) at thedesired depth to target the nerve endings, and the vector solution thenmay be injected as a single bolus dose, or slowly through an infusionpump (0.01 to 1 mL/min). More specifically in an exemplary configurationwherein a renal nerve plexus is targeted to address hypertension viaintraparenchymal injection into one or more of the kidneys, the numberof injections and dose of virus injected to the kidneys may beapproximated from the primate viral retrograde transport study performedby Towne et al (Gene Ther. 2010 January; 17(1):141-6), incorporated byreference in its entirety herein. Such protocols have been shown toachieve efficient retrograde transport following injection of 1 mLsaline solution containing 1.3×10¹² viral genomes of AAV6 into a tissueof approximately 30 cm³ volume. Considering that the total kidneyparenchyma has a volume of approximately 150 cm³, it is possible toachieve efficient retrograde transport using 5 mL saline solutioncontaining approximately 6.5×10¹² viral genomes of the desired vector.This 5 mL may be injected over multiple sites to evenly disperse thevector throughout the volume of the kidney parenchyma. For example, inone embodiment, about 20 injections of 0.25 mL containing 3.25×10¹⁰ vgof vector may be made at approximately equidistant sites throughout theparenchyma of the kidney for successful transfection. These ranges areillustrative, and doses are tested for each virus-promoter-opsinconstruct pairing them with the targeted neurons.

Tissue structures such as the wall of the stomach may also be directlytargeted for viral injection. For example, in one embodiment it may bedesirable to inject the stomach wall to target the stretch receptors toaddress obesity-related clinical challenges. In such an embodiment,after creating an access pathway, such as a small laparoscopic incisionto allow laparoscopic tools (camera, needle, tools, etc.) to approachthe stomach wall, a needle may be inserted into the stomach wall in thevicinity of the nerve endings for the stretch receptors. The needle maybe guided into the pertinent anatomy using the available laparoscopicimaging tools, such as one or more cameras, ultrasound, fluoroscopy, orthe like. The pertinent vector solution may injected through the needlewhere it may diffuse throughout the tissue and be taken up by the neuralterminals (i.e. stretch and chemical afferent fiber nerve endings). Thevector solution may be injected as a single bolus dose, or slowlythrough an infusion pump (0.01 to 1 mL/min). Once taken up by suchneural terminals, the vector may be retrogradely transported to thepertinent neural cell body or bodies along the length of the pertinentaxons. The number of injections and dose of virus injected to thestomach wall may be approximated from the primate viral retrogradetransport study performed by Towne et al (Gene Ther. 2010 January;17(1):141-6), incorporated by reference herein in its entirety. Thisstudy demonstrated efficient retrograde transport following injection of1 mL saline solution containing 1.3×10¹² viral genomes of AAV6 into amuscle of approximately 30 cm³ volume. Considering that the stomach wallhas an average thickness of approximately 4 mm and a surface area ofapproximately 150 cm² (total targeted tissue structure volume of about60 cm³), efficient retrograde transport may be achieved using 2 mLsaline solution containing approximately 3×10¹² viral genomes of thedesired vector. This 2 mL may be injected over multiple sites to evenlydisperse the vector over the surface area of stomach wall. For example,about 20 injections of 0.1 mL containing 1.3×10¹¹ vg of vector may beconducted for each 7.5 cm² area of stomach wall. These titers andinjection volumes are illustrative examples and are specificallydetermined for each viral construct-target neuron pairing.

In other embodiments, nerve fibers may be targeted by direct injection(i.e., injection into the nerve itself). This approach, which may betermed “intrafascicular” or “intraneural” injection, involves placing aneedle into the fascicle of a nerve bundle. Intrafascicular injectionsare an attractive approach because they allow specific targeting thoseneurons which may innervate a relatively large target (e.g., fibersacross entire kidney, fibers across entire dermatome of skin, fibersacross entire stomach wall) with one injection (e.g., before the fibersenter the tissue and anatomically bifurcate). The pertinent vectorsolution may be injected through the needle where it may diffusethroughout the entire nerve bundle (10 to 1000's of axon fibers). Thevector may then enter the individual axon fibers through active(receptor-mediated) or passive (diffusion across intact membranes ortransiently disrupted membranes) means. Once it has entered the axon,the vector may be delivered to the cell body via retrograde transportmechanisms, as described above. The number of injections and dose ofvirus injected to the nerve are dependent upon the size of the nerve,and can be extrapolated from successful transduction studies. Forexample, injection of the sciatic nerve of mice (approximately 0.3 mmdiameter) with 0.002 mL saline containing 1×10⁹ vg of AAV has been shownto result in efficient transgene delivery to sensory neurons involved inpain sensing. Likewise, injection of the sciatic nerve of rats (1 mmdiameter) with 0.010 mL saline containing 1-2×10¹⁰ vg of AAV has alsoachieved desirable transfection results. The trigeminal nerve in humansis 2 mm in diameter, and through extrapolation of the data from thesepertinent studies, the trigeminal nerve may be transfected toefficiently deliver a transgene to these pertinent pain neurons using adirect injection of 0.05 mL saline containing 4×10¹⁰-1×10¹⁴ vg of AAVinto the trigeminal bundle. These titers and injection volumes areillustrative examples and are specifically determined for each viralconstruct-target neuron pairing.

The protocol for nerve injections will vary depending upon the target.Superficial nerves may be targeted by making an incision through theskin, and then exposing the nerve through separation of muscles, fasciaand tendons. Deeper nerves (i.e., outside of the abdominal and thoraciccavity—such as the pudendal nerve) may be targeted throughultrasound-guided surgical intervention. Nerves in the abdominal cavitymay be targeted through laparoscopic surgical approaches wherein one ormore small incisions may be made through the skin and other structures(such as the abdominal wall) to allow insertion of the surgicalapparatus (camera, needle, tools, etc.) to a position adjacent theanatomy of interest. The needle may be guided into the nerve (asvisualized through the camera and other available imaging systems, suchas ultrasound, fluoroscopy, radiography, etc.). In all cases, the vectorsolution may be injected as a single bolus dose, or slowly through aninfusion pump (0.001 to 0.1 mL/min).

In one particular example, the gastric and hepatic branches of the vagusnerve may be directly injected to control satiety. In such anembodiment, laparoscopic surgery may be performed to target the gastricand hepatic branches of the vagus nerve that lie adjacent to theesophagus and innervate the stomach with the clinical goal being toinfect the fibers of the afferent stretch receptors in the stomach wallvia direct injection of vector material into these vagus nerve branches,preferably as facilitated by one or more imaging technologies asdescribed above.

In another particular example of intraneural injection, nociceptivefibers of the trigeminal nerve may be directly injected to addressneuropathic pain symptoms, as briefly described above. In oneembodiment, the trigeminal nerve may be directly injected with an AAVvector solution either through exposure of the nerve or through the skinvia ultrasound guidance. Once in the nerve fascicle, the vector isconfigured to preferentially enter the non-myelinated orpoorly-myelinated fibers that correspond to those cells mediating pain.

In another particular example of intraneural injection, the sciaticnerve may be injected with an AAV vector solution either throughexposure of the nerve or through the skin via ultrasound guidance. Thevector may be configured such that once it accesses the nerve fascicle,it preferentially enters the sensory neurons or motor neuronsresponsible for the symptoms of spasticity.

In another particular example of intraneural injection, the cervicalvagus nerve may be injected with an AAV vector solution through exposureof the nerve in the neck. Once in the nerve fascicle, the vector may beconfigured to preferentially enter the relevant nerve fibers that arethe mediators of the therapeutic effect of electrical vagus nervestimulation for epilepsy.

In another particular example of intraneural injection, the cervicalvagus nerve may be injected with an AAV vector solution through exposureof the nerve in the neck. Once in the nerve fascicle, the vector may beconfigured to preferentially enter the relevant nerve fibers that arethe mediators of the therapeutic effect of vagus electric nervestimulation for depression.

As mentioned above, injection into the ganglion may be utilized totarget the neural cell bodies of peripheral nerves. Ganglia consist ofsensory neurons of the peripheral nervous system, as well as autonomicneurons of the parasympathetic and sympathetic nervous system. A needlemay be inserted into the ganglion which contains the cell bodies and avector solution injected through the needle, where it may diffusethroughout the tissue and be taken up by the cell bodies (100s to 1000sof cells). In one embodiment, a dose of approximately 0.1 mL salinecontaining from 1×10¹¹ vg to 1×10¹⁴ vg of AAV may be used per ganglion.There are different types of ganglia that may be targeted. Dorsal rootganglion of the spinal cord may be injected in a similar method that isused during selective dorsal rhizotomy (i.e. injection via theintrathecal subarachnoid space of the spinal cord), except rather thancutting the nerves, the dorsal root ganglia may be injected. Otherganglia not in the abdominal cavity, such as the nodose ganglion of thevagus nerve, may be targeted by making an incision through the skin, andthen exposing the ganglia through separation of muscles, fascia andtendons. Ganglia in the abdominal cavity, such as the ganglia of therenal plexus, may be injected through laparoscopic techniques, whereinone or more small incisions may be made through the skin and abdominalwall to allow insertion of the surgical apparatus (camera, needle,tools, etc.) to locations facilitating access and imaging of thepertinent targeted tissue. The needle may be guided into the ganglia (asvisualized through a camera or other imaging device, such as ultrasoundor fluoroscopy). In all cases, the vector solution may be injected as asingle bolus dose, or slowly through an infusion pump (0.001 to 0.1mL/min). These ranges are illustrative, and doses are tested for eachvirus-promoter-opsin construct pairing them with the targeted neurons.

In one particular example of ganglion injection, the dorsal root gangliamediating clinical neuropathic pain may be injected with an AAV vectorsolution, preferably containing an AAV vector that has tropism for cellbody.

In another particular example of ganglion injection, the dorsal rootganglia mediating undesired muscular spasticity may be injected with anAAV vector solution. An AAV vector that has tropism for cell body may beused towards this goal.

In another particular example of ganglion injection, the nodose ganglionmay be exposed and injected with an AAV vector solution to addressclinical epilepsy symptoms. An AAV vector that has tropism for cell bodymay be used towards this goal to infect specifically the afferent cellsthat are thought to mediate the therapeutic effect of vagus nervestimulation. In one embodiment the AAV vector is injected into a cellbody. In another embodiment the AAV vector is injected into a targettissue and retrogradely transported to a cell body. Embodiments of theinvention include optical stimulation at a cell body or along an axon.

In another particular example of ganglion injection, the nodose ganglionmay be exposed and injected with an AAV vector solution. An AAV vectorthat has tropism for cell body may be used towards this goal to infectspecifically the afferent cells that are thought to mediate thetherapeutic effect of vagus nerve stimulation.

In another particular example of ganglion injection, ganglion of therenal plexus may be injected for hypertension treatment. Laparoscopicsurgery may be performed to target the ganglion of the renal plexus.Ganglia adjacent to the kidneys and on the renal arteries may beidentified and then injected individually and directly with oneobjective being to infect the cell bodies of the renal plexus efferentneurons.

As noted above, direct injection of vascular structures such as arterywalls may also be utilized to deliver genetic material for optogenetictherapy. For example, in one embodiment, portions of one or more of therenal arteries may be directly injected to infect the nearby renalplexus to address hypertension (the renal arteries are surrounded by aneural plexus that mediates control of blood pressure via the kidneys,as described in further detail below). A small incision may be madethrough the skin and abdominal wall to allow insertion of a laparoscopicsurgical system (camera, needle, tools, etc.). A needle may be guidedinto the renal plexus (as visualized through the camera or other imagingdevice). The needle may be placed in multiple sites around thecircumference of the renal arteries. The vector solution is injectedthrough the needle where it may diffuse throughout the arterial wall andbe taken up through by the adjacent renal plexus nerve fibers viadiffusion across intact membranes (or transiently disrupted membranes).In all cases, the vector solution may be injected as a single bolusdose, or slowly through an infusion pump (0.001 to 0.1 mL/min). Multipleinjections of a dose of approximately 0.1 mL saline containing 1×10¹¹ vgof AAV may be used at different sites around the circumference andlength of the renal arteries (in the vicinity of the renal plexus), withthe goal being to infect the axons of the renal plexus efferent neurons.The amount of virus is illustrative of such transfection but optimumdosing will vary depending on the target neuron paired with a specificvirus-promoter-opsin construct.

Finally, as noted above, internal topical injection or application to atissue structure surface may be utilized to deliver genetic material foroptogenetic therapy. Recombinant vectors are capable of diffusingthrough membranes and infecting neural nerve endings following suchtopical application or exposure. Examples are the infection of sensoryfibers following topical application on skin, which has been shown inpain treatment studies. Likewise, efficacy of topical application ofviral vectors has been increased using vector solutions suspended ingels. In one embodiment, a vector may be suspended in a gel and applied(e.g., swabbed, painted, injected, or sprayed) to the surface of tissuesthat have high densities of targeted superficial nerve fibers. With suchembodiment, vectors will diffuse through the gel and infect nerve fibersvia diffusion across intact neural fiber membranes. Internal topicalapplication may be achieved using laparoscopic techniques, wherein oneor more small incisions may be made through the skin and other pertinenttissue structures (such as the abdominal wall) to allow insertion of thesurgical apparatus (camera, needle, tools, etc.). A needle may be guidedinto the target tissue (as visualized through the camera or otherimaging devices). In all cases, the vector may be mixed with the gel(e.g. the product sold under the tradename “KY Jelly” by Johnson &Johnson Corporation) and then sprayed onto, painted onto, or injectedout upon the surface of the pertinent tissue. A dose of approximately0.1 mL saline containing 1×10¹¹ vg of AAV may be used to cover each 1cm² area. These ranges are illustrative, and doses are tested for eachvirus-promoter-opsin construct pairing them with the targeted neurons.

In one particular example of topical application, afferent nerve fibersof the stomach wall may be targeted and infected to address a clinicalsatiety challenge. Laparoscopic surgical techniques may be utilized totarget the superficial nerve fibers that project into the stomach wallfrom the gastric and hepatic vagus nerve, with the clinical goal beingto infect the fibers of the afferent stretch receptors in the stomachwall to facilitate optogenetic induction of satiety. Upon successfullaparoscopic access, a solution or gel may be applied to infect thetargeted nerve tissues.

In another particular example of topical application, hypertension maybe addressed by topical application of vector solution or gel to therenal plexus from a laparoscopic approach to achieve transfer ofoptogenetic material to the pertinent nerves. Laparoscopic surgery maybe performed to target the surface of the renal plexus directly. Therenal arteries and kidneys may be identified using one or more imagingdevices (such as a camera, ultrasound, fluoroscopy, radiography, etc.)and then the vector may be applied directly and topically at multiplesites to cover as much of the available nerve plexus surface aspossible, the goal being to infect the axons of the renal plexusefferent neurons.

Prior to implantation or injection, patients may be started on a clearliquid diet on postoperative day 1. Following induction with generalendotracheal anesthesia and the administration of a broad-spectrumprophylactic intravenous antibiotic and/or cystoscopy and/or retrogradepyelography may be performed, and a long indwelling optical therapeuticdevice, such as an applicator, and/or delivery segments, and/or ahousing may be passed. A Foley catheter and orogastric tube may also beplaced, by way of non-limiting examples. The patient may be placed in a45-degree lateral decubitus position and secured to the operating table.Insufflation may be performed, for example, through a Veress needle, andlaparoscopic ports may be passed into the peritoneal cavity. Theipsilateral colon may be reflected, and the proximal ureter and renalpelvis may be identified and fully mobilized. Dissection of an extensivelength of proximal ureter may be avoided in an attempt to preserve anycollateral vascular supply. If a crossing vessel is present, fibroticbands between the vessels and collecting system may be divided to gainunobstructed access to the renal pelvis beyond the ureteropelvicjunction (UBJ). The catheter may be cleared at this point, and theoptical activator and/or delivery segments and/or housing thenintroduced into the renal pelvis. To close, a 5-mm closed suction drain,for example, may be placed through a posterior stab incision into theperinephric space adjacent to the UBJ. Hemostasis may be confirmed, theCO₂ may be evacuated, and the port sites may be closed. The orogastrictube may be removed prior to catheter removal.

The renal plexus (52) generally resides around the renal arteryunderneath a layer of renal fascia (64), as described in the followingtable.

Distance From Outer Artery Cumulative % Surface (mm) of Nerves 0.5 9 125 1.5 51 2 99 2.5 100

The renal artery surface irradiance parameters are different from thoseof the target threshold irradiance because there are very few nerves inthe outermost portions of the renal artery, most being located muchcloser to the intima between 1.5-2.0 mm beneath the outer surface. Tocompensate for irradiance diminution due to optical scattering, theirradiance delivered to the tissue surface is higher than that requiredfor optical activation at the target depth. The scattering cross-sectionmonotonically decreases with wavelength. As an illustrative example,although not rigorously analytical, reasonable approximations for therequired surface irradiance relative to that required at the targetdepths shown in the above table grouped into spectral bands may be made,and are listed in the following table.

Spectral Irradiance Region Increase 450-480 nm 21 481-530 nm 15 531-560nm 10 561-600 nm 6

The optical parameters required to effectively activate specificoptogenetic targets are listed in the following table.

Renal Pulse Surface Duration Duty Irradiance OPSIN (ms) Cycle (%)(mW/mm²) NpHR 0.1-10 50-100 5-50 eNpHR 3.0 0.1-10 50-100 5-50 ARCH 3.00.1-10 50-100 10-100 Mac 3.0 0.1-10 50-100 15-150 ChR2   2-20 5-5020-200 VChR1   2-20 5-50 15-150 CatCH   2-20 5-50 20-200 C1V1   2-205-50 20-200 SFO 0.1-10 0.1-1   15-150 SSFO 0.1-10 0.1-1   15-150

For Renal Nerve Inhibition, photosensitivity may be preferred overspeed, or response time, of the opsin. Therefore, ChR2 opsins utilizingthe C123S, and/or C128A, and/or C128S, and/or the D156A mutations, suchas SFO and/or SSFO, may be used, as they may comprise the desiredrelatively high level of light sensitivity, although potentially withrelatively lower temporal resolution. Alternately, mutations atanalogous positions to ChR2 C123S, and/or C128A, and/or C128S, and/orthe D156A in other may be added to other opsins. Alternately, the SFOand/or SSFO variants may be used with a 2-color illumination system,such as that shown in the exemplary system of FIG. 16, wherein light ofa first color (such as blue light) may be used to activate the opsin,and light of a second wavelength (such as yellow light) may be used tode-activate the opsin, such as may be performed in an exemplaryconfiguration for depolarization (hyperstimulation) block. Alternately,in a similar fashion, the C1V1 E122T, E162T, and/or E122T/E162T variantsmay comprise the desired relatively high level of light sensitivity.Alternately, ChiEF, and/or ChrFR, and/or ChrGr, including those withmutations analogous to the ChR2 SFO and/or SSFO mutations may be used ina configuration for depolarization (hyperstimulation) block.Alternately, opsins, such as, but not limited to, NpHR 3.0, and/or ARCH3.0, may be for direct inhibition. An inhibitory opsin may be selectedfrom those listed in FIG. 62J (FIGS. 62J-1 and 62J-2), by way ofnon-limiting examples. A stimulatory opsin may be selected from thoselisted in FIG. 62J (FIGS. 62J-1 and 62J-2, by way of non-limitingexamples. An opsin may be selected from the group consisting ofOpto-β2AR or Opto-α1AR, by way of non-limiting examples.

Any of the slab-type, and/or cuff-type, and/or spiral-type, and theirrespective systems herein described and shown in FIGS. 5, 8-14, and17-28, or elsewhere herein, may be used to deliver therapeutic light tothe exterior of the renal artery in order to reach the optogenetictargets within the renal nerve plexus. However, the blood residing inthe renal artery is also an avid absorber of visible light and mayrender moot the light-recycling elements of those applicators for lightthat traverses the blood-filled lumen. It should be noted, however, thatlight-recycling still serves to increase the overall system efficiencyfor light that escapes otherwise and nominally avoids optical contactwith blood.

FIG. 63 shows an alternate embodiment, where an Applicator A is similarto that shown in FIGS. 25-27, being further configured to comprise aweb-like substrate SUB configured to lie on the outer surface of thekidney to illuminate the renal pelvis. In this exemplary embodiment,Applicator A comprises Substrate SUB, which is configured as a flexibleweb structure that may allow for it to be draped onto the target tissue,in this case a RENAL PELVIS of a KIDNEY. As has been described earlier,Delivery Segments DSx connect Applicator A to at least a portion of asystem controller, shown here as Housing H. An illustrative example of aweb-like Substrate SUB is shown in Kim, et al, “Waterproof AlInGaPoptoelectronics on stretchable substrates with applications inbiomedicine and robotics” Nature Materials 9, 929-937 (2010), which isincorporated herein by reference in its entirety. Anchoring Features AFxare also shown, and may provide means for the applicator to beaffirmatively affixed and located on the target tissue. AnchoringFeatures AF may be, by way of non-limiting examples; tines, barbs,and/or closure holes (as described elsewhere herein).

Referring back to FIG. 1, after delivery of the polynucleotide to thetargeted neuroanatomy (6), an expression time period generally isrequired to ensure that sufficient portions of the targeted neuroanatomywill express the light-responsive opsin protein upon exposure to light(8). This waiting period may, for example, comprise a period of betweenabout 1 month and 4 months. After this period of time, light may bedelivered to the targeted neuroanatomy to facilitate the desiredtherapy. Such delivery of light may take the form of many differentconfigurations, including transcutaneous configurations, implantableconfigurations, configurations with various illumination wavelengths,pulsing configurations, tissue interfaces, etc., as described below infurther detail.

Referring to FIGS. 2A and 2B, both of which are end views showing across sectional anatomical face (N) and a cross sectional view of atreatment assembly which in orthogonal view may, for example, berectangular, trapezoidal, or elliptical (i.e., so that it may provide asufficient area of exposure to the anatomy N when in contact), a matrixof needles or needle-like injection structures (22) may be utilized toinject a vector solution or gel in a circumferential manner around anerve (20), nerve bundle, vessel surrounded by nerve fibers, or othersomewhat cylindrical targeted anatomic structure into which injection isdesired. As shown in FIG. 2A, a flexible or deformable housing (24) mayfeature a bending spine member (26) configured to bias the housing intoa cylindrical (i.e., like a cuff), arcuate, helical, or spiral shapewithout other counterbalancing loads. For example, the bending spinemember may comprise a superalloy such as Nitinol, which may beconfigured through heat treatment to be pre-biased to assume suchcylindrical, arcuate, helical, or spiral shape. The depicted embodimentof the housing (24) also features two embedded bladders—an injectionbladder (36) which is fluidly coupled between the matrix of injectionmembers (22) and an injection reservoir by a fluid conduit (16) such asa tube or flexible needle, and a mechanical straightening bladder (38),which is fluidly coupled to a straightening pressure reservoir (14) by afluid conduit (18) such as a tube or flexible needle. Preferably bothfluid conduits (16, 18) are removably coupled to the respective bladders(36, 38) by a removable coupling (32, 34) which may be decoupled bymanually pulling the conduits (16, 18) away from the housing (24). Thehousing (24) may be inserted, for example, through a port in alaparoscopic tool, cannula, or catheter and inserted to a position asshown in FIG. 2A with the straightening bladder (38) fully pressurizedto bias the housing into the shown flat condition with the ends rotateddownward (28) due to the pressure applied through the straighteningpressure reservoir (14), for example using an operatively coupledsyringe or controllable pump, and functionally delivered through theassociated conduit (18). With the straightened housing (24) in adesirable position relative to the targeted anatomic structure (20),preferably as confirmed using one or more visualization devices such asa laparoscopic camera, ultrasound transducer, fluoroscopy, or the like,the pressure within the straightening pressure reservoir (14) may becontrollably decreased (for example, in one embodiment, the associatedconduit 18 may simply be disconnected from the coupling 34) to allow theends of the housing (24) to flex and rotate (30) up and around theanatomical structure (20) due to the now un-counterbalanced bendingloads applied by the pre-bending-biased bending spine member (26). FIG.2B depicts the ends starting to rotate up and around (30) the anatomicalstructure (20). With complete rotation, the flexible housing preferablywill substantially surround at least a portion of the anatomicalstructure (20) in an arcuate, cuff, helical, or spiral configurationwith the matrix of needles (22) interfaced directly against the outersurface of the anatomical structure (20), after which the pressurewithin the injection reservoir (12) may be controllably increased, forexample using an infusion pump or syringe, to inject the anatomicalstructure (20) with the desired solution or gel. In one embodiment, itmay be desirable to leave the housing in place as a prosthesis; inanother embodiment it may be desirable to remove the housing aftersuccessful injection. In the former scenario, in one variation, thehousing may also comprise a light delivery interface, such as isdescribed below (i.e., in addition to a bending spine 26, astraightening bladder 38, an injection bladder 36, and a matrix ofneedles 22, the housing 24 may also comprise one or more light deliveryfibers, lenses, and the like, as described below, to facilitate lighttherapy after injection of the pertinent genetic material). In thelatter scenario, wherein the housing is to be removed after injection,the straightening pressure conduit (18) will remain coupled to thestraightening bladder (38) so that after injection has been completed,the pressure within the straightening reservoir (14) may again becontrollably increased, thereby rotating (28) the housing back out intoa flat configuration as shown in FIG. 2A such that it may then beremoved away from the subject anatomy (20). In one embodiment, thematrix of needles (22) may reside upon a movable or flexible membrane orlayer relative to the supporting housing (24), and may be biased torecede inward toward the housing (24) when the injection pressure is notheightened, and to become more prominent relative to the supportinghousing (24) when the injection pressure is increased; in other words,to assist with delivery and retraction (i.e., so that the housing 24 maybe moved around relative to other nearby tissues without scratching,scraping, injuring, or puncturing such tissues without intention), whenthe injection pressure is relatively low, the injection structures maybe configured to become recessed into the housing. It may also bedesirable to have the matrix of needles (22) retract subsequent toinjection to generally prevent tissue trauma upon exit of the housing(24) in the event that the housing (24) is to be removed, or to preventfibrous tissue encapsulation of the targeted tissue structure (4) whichmay be associated with or accelerated by relatively abrasive orindwelling foreign body presence. Indeed, in one embodiment wherein thehousing (24) is to remain in place (for example, as anillumination/light applicator platform), the matrix of needles (22) maycomprise a bioresorbable material such as PLGA, which is commonlyutilized in surgery for its resorbable qualities and may be configuredto dissolve and/or resorb away within a short time period afterinjection has been completed.

Referring to FIG. 3, a suitable light delivery system comprises one ormore applicators (A) configured to provide light output to the targetedtissue structures. The light may be generated within the applicator (A)structure itself, or within a housing (H) that is operatively coupled tothe applicator (A) via one or more delivery segments (DS). The one ormore delivery segments (DS) serve to transport, or guide, the light tothe applicator (A) when the light is not generated in the applicatoritself. In an embodiment wherein the light is generated within theapplicator (A), the delivery segment (DS) may simply comprise anelectrical connector to provide power to the light source and/or othercomponents which may be located distal to, or remote from, the housing(H). The one or more housings (H) preferably are configured to servepower to the light source and operate other electronic circuitry,including, for example, telemetry, communication, control and chargingsubsystems. External programmer and/or controller (P/C) devices may beconfigured to be operatively coupled to the housing (H) from outside ofthe patient via a communications link (CL), which may be configured tofacilitate wireless communication or telemetry, such as viatranscutaneous inductive coil configurations, between the programmerand/or controller (P/C) devices and the housing (H). The programmerand/or controller (P/C) devices may comprise input/output (I/O) hardwareand software, memory, programming interfaces, and the like, and may beat least partially operated by a microcontroller or processor (CPU),which may be housed within a personal computing system which may be astand-alone system, or be configured to be operatively coupled to othercomputing or storage systems.

Referring to FIGS. 4A and 4B, as described above, various opsin proteinconfigurations are available to provide excitatory and inhibitoryfunctionality in response to light exposure at various wavelengths. FIG.4A (1000) depicts wavelength vs. activation for three different opsins;FIG. 4B (1002) emphasizes that various opsins also have time domainactivation signatures that may be utilized clinically; for example,certain step function opsins (“SFO”) are known to have activations whichlast into the range of 30 minutes after stimulation with light.

Referring to FIG. 4C (FIGS. 4C-1 and 4C-2) (1004), a variety oflight-emitting diodes (LED) are commercially available to provideillumination at relatively low power with various wavelengths. Asdescribed above in reference to FIG. 3, in one embodiment, light may begenerated within the housing (H) and transported to the applicator (A)via the delivery segment (DS). Light may also be produced at or withinthe applicator (A) in various configurations. The delivery segments (DS)may consist of electrical leads or wires without light transmittingcapability in such configurations. In other embodiments, light may bedelivered using the delivery segments (DS) to be delivered to thesubject tissue structures at the point of the applicator (A), or at oneor more points along the deliver segment (DS) itself (for example, inone case the DS may be a fiber laser). Referring again to FIG. 4C (FIGS.4C-1 and 4C-2) (1004), an LED (or alternatively, “ILED”, to denote thedistinction between this inorganic system and Organic LEDs) typically isa semiconductor light source, and versions are available with emissionsacross the visible, ultraviolet, and infrared wavelengths, withrelatively high brightness. When a light-emitting diode isforward-biased (switched on), electrons are able to recombine withelectron holes within the device, releasing energy in the form ofphotons. This effect is called electroluminescence and the color of thelight (corresponding to the energy of the photon) is determined by theenergy gap of the semiconductor. An LED is often small in area (lessthan 1 mm²), and integrated optical components may be used to shape itsradiation pattern. In one embodiment, for example, an LED variationmanufactured by Cree Inc. and comprising a Silicon Carbide deviceproviding 24 mW at 20 mA may be utilized as an illumination source.

Organic LEDs (or “OLED”s) are light-emitting diodes wherein the emissiveelectroluminescent layer is a film of organic compound that emits lightin response to an electric current. This layer of organic semiconductormaterial is situated between two electrodes, which can be made to beflexible. At least one of these electrodes may be made to betransparent. The nontransparent electrode may be made to serve as areflective layer along the outer surface on an optical applicator, aswill be explained later. The inherent flexibility of OLEDs provides fortheir use in optical applicators such as those described herein thatconform to their targets or are coupled to flexible or movablesubstrates, as described above in reference to FIGS. 2A-2B, and infurther detail below. It should be noted, however, due to theirrelatively low thermal conductivity, OLEDs typically emit less light perarea than an inorganic LED.

Other suitable light sources for embodiments of the inventive systemsdescribed herein include polymer LEDs, quantum dots, light-emittingelectrochemical cells, laser diodes, vertical cavity surface-emittinglasers, and horizontal cavity surface-emitting lasers.

Polymer LEDs (or “PLED”s), and also light-emitting polymers (“LEP”),involve an electroluminescent conductive polymer that emits light whenconnected to an external voltage. They are used as a thin film forfull-spectrum color displays. Polymer OLEDs are quite efficient andrequire a relatively small amount of power for the amount of lightproduced.

Quantum dots (or “QD”) are semiconductor nanocrystals that possessunique optical properties. Their emission color may be tuned from thevisible throughout the infrared spectrum. They are constructed in amanner similar to that of OLEDs.

A light-emitting electrochemical cell (“LEC” or “LEEC”) is a solid-statedevice that generates light from an electric current(electroluminescence). LECs may be usually composed of two electrodesconnected by (e.g. “sandwiching”) an organic semiconductor containingmobile ions. Aside from the mobile ions, their structure is very similarto that of an OLED. LECs have most of the advantages of OLEDs, as wellas a few additional ones, including:

-   -   The device does not depend on the difference in work function of        the electrodes. Consequently, the electrodes can be made of the        same material (e.g., gold). Similarly, the device can still be        operated at low voltages;    -   Recently developed materials such as graphene or a blend of        carbon nanotubes and polymers have been used as electrodes,        eliminating the need for using indium tin oxide for a        transparent electrode;    -   The thickness of the active electroluminescent layer is not        critical for the device to operate, and LECs may be printed with        relatively inexpensive printing processes (where control over        film thicknesses can be difficult).

Semiconductor Lasers are available in a variety of output colors, orwavelengths. There are a variety of different configurations availablethat lend themselves to usage in the present invention, as well. Indiumgallium nitride (In_(x)Ga_(1-x)N, or just InGaN) laser diodes have highbrightness output at both 405, 445, and 485 nm, which are suitable forthe activation of ChR2. The emitted wavelength, dependent on thematerial's band gap, can be controlled by the GaN/InN ratio; violet-blue420 nm for 0.2In/0.8Ga, and blue 440 nm for 0.31n/0.7Ga, to red forhigher ratios and also by the thickness of the InGaN layers which aretypically in the range of 2-3 nm.

A laser diode (or “LD”) is a laser whose active medium is asemiconductor similar to that found in a light-emitting diode. The mostcommon type of laser diode is formed from a p-n junction and powered byinjected electric current. The former devices are sometimes referred toas injection laser diodes to distinguish them from optically pumpedlaser diodes. A laser diode may be formed by doping a very thin layer onthe surface of a crystal wafer. The crystal may be doped to produce ann-type region and a p-type region, one above the other, resulting in ap-n junction, or diode. Laser diodes form a subset of the largerclassification of semiconductor p-n junction diodes. Forward electricalbias across the laser diode causes the two species of chargecarrier—holes and electrons—to be “injected” from opposite sides of thep-n junction into the depletion region. Holes are injected from thep-doped, and electrons from the n-doped, semiconductor. (A depletionregion, devoid of any charge carriers, forms as a result of thedifference in electrical potential between n- and p-type semiconductorswherever they are in physical contact.) Due to the use of chargeinjection in powering most diode lasers, this class of lasers issometimes termed “injection lasers” or “injection laser diodes” (“ILD”).As diode lasers are semiconductor devices, they may also be classifiedas semiconductor lasers. Either designation distinguishes diode lasersfrom solid-state lasers. Another method of powering some diode lasers isthe use of optical pumping. Optically Pumped Semiconductor Lasers (or“OPSL”) use a III-V semiconductor chip as the gain media, and anotherlaser (often another diode laser) as the pump source. OPSLs offerseveral advantages over ILDs, particularly in wavelength selection andlack of interference from internal electrode structures. When anelectron and a hole are present in the same region, they may recombineor “annihilate” with the result being spontaneous emission—i.e., theelectron may re-occupy the energy state of the hole, emitting a photonwith energy equal to the difference between the electron and hole statesinvolved. (In a conventional semiconductor junction diode, the energyreleased from the recombination of electrons and holes is carried awayas phonons, i.e., lattice vibrations, rather than as photons.)Spontaneous emission gives the laser diode below lasing thresholdsimilar properties to an LED. Spontaneous emission is necessary toinitiate laser oscillation, but it is one among several sources ofinefficiency once the laser is oscillating. The difference between thephoton-emitting semiconductor laser and conventional phonon-emitting(non-light-emitting) semiconductor junction diodes lies in the use of adifferent type of semiconductor, one whose physical and atomic structureconfers the possibility for photon emission. These photon-emittingsemiconductors are the so-called “direct bandgap” semiconductors. Theproperties of silicon and germanium, which are single-elementsemiconductors, have bandgaps that do not align in the way needed toallow photon emission and are not considered “direct.” Other materials,the so-called compound semiconductors, have virtually identicalcrystalline structures as silicon or germanium but use alternatingarrangements of two different atomic species in a checkerboard-likepattern to break the symmetry. The transition between the materials inthe alternating pattern creates the critical “direct bandgap” property.Gallium arsenide, indium phosphide, gallium antimonide, and galliumnitride are all examples of compound semiconductor materials that may beused to create junction diodes that emit light.

Vertical-cavity surface-emitting lasers (or “VCSEL”s) have the opticalcavity axis along the direction of current flow rather thanperpendicular to the current flow as in conventional laser diodes. Theactive region length is very short compared with the lateral dimensionsso that the radiation emerges from the surface of the cavity rather thanfrom its edge as shown in the figure. The reflectors at the ends of thecavity are dielectric mirrors made from alternating high and lowrefractive index quarter-wave thick multilayer. VCSELs allow formonolithic optical structures to be produced.

Horizontal cavity surface-emitting lasers (or “HCSEL”s) combine thepower and high reliability of a standard edge-emitting laser diode withthe low cost and ease of packaging of a vertical cavity surface-emittinglaser (VCSEL). They also lend themselves to use in integrated on-chipoptronic, or photonic packages.

The irradiance required at the neural membrane in which the optogeneticchannels reside is on the order of 0.05-2 mW/mm² and depends uponnumerous elements, such as opsin channel expression density, activationthreshold, etc. A modified channelrhodopsin-2 resident within a neuronmay be activated by illumination of the neuron with green or blue lighthaving a wavelength of between about 420 nm and about 520 nm, and in oneexample about 473 nm, with an intensity of between about 0.5 mW/mm² andabout 10 mW/mm², such as between about 1 mW/mm² and about 5 mW/mm², andin one example about 2.4 mW/mm². Although the excitation spectrum may bedifferent, similar exposure values hold for other opsins, such as NpHR,as well. Because most opsin-expressing targets are contained within atissue or other structure, the light emitted from the applicator mayneed to be higher in order to attain the requisite values at the targetitself. Light intensity, or irradiance, is lost predominantly due tooptical scattering in tissue, which is a turbid medium. There is alsoparasitic absorption of endogenous chromophores, such as blood, that mayalso diminish the target exposure. Because of these effects, theirradiance range required at the output of an applicator is, for most ofthe cases described herein, between 1-100 mW/mm². Referring to FIG. 5,experiments have shown, for example, that for the single-sided exposureof illumination (I) from an optical fiber (OF) of a 1 mm diameter nervebundle (N), the measured response (in arbitrary units) vs. irradiance(or Light Power Density, in mW/mm²) is asymptotic, as shown in the graphdepicted in FIG. 6 (1006). There is not appreciable improvement beyond20 mW/mm² for this specific configuration of opsin protein, expressiondensity, illumination geometry, and pulse parameters. However, we mayuse this result to scale the irradiance requirements to other targetswith similar optical properties and opsin protein expression densities.The data in FIG. 6 (1006) may be used in a diffusion approximationoptical model for neural materials, where the irradiance (I) obeys thefollowing relation, I=I_(o)e^(−(Qμz)). The resulting expression fitswell with the following experimental data, and the result of this isgiven in the plot of FIG. 7 (1008). The details are further discussedbelow.

The optical penetration depth, δ, is the tissue thickness that causeslight to attenuate to e⁻¹ (˜37%) of its initial value, and is given bythe following diffusion approximation.

${\delta = \frac{1}{\sqrt{3\mu_{a}\mu_{s}^{\prime}}}},$

where μ_(a) is the absorption coefficient, and μ_(s′) is the reducedscattering coefficient. The reduced scattering coefficient is a lumpedproperty incorporating the scattering coefficient μ_(s) and theanisotropy g: μ_(s)′=μ_(s)(1−g) [cm⁻¹]. The purpose of μ_(s)′ is todescribe the diffusion of photons in a random walk of step size of1/μ_(s)′ [cm] where each step involves isotropic scattering.

Such a description is equivalent to description of photon movement usingmany small steps 1/μ_(s) that each involve only a partial deflectionangle θ, if there are many scattering events before an absorption event,i.e., μ_(a)<<μ_(s)′. The anisotropy of scattering, g, is effectively theexpectation value of the scattering angle, θ. Furthermore, the“diffusion exponent,” μ_(eff), is a lumped parameter containing ensembleinformation regarding the absorption and scattering of materials,μ_(eff)=Sqrt(3μ_(a)(μ_(a)+μ_(s′)). The cerebral cortex constitutes asuperficial layer of grey matter (high proportion of nerve cell bodies)and internally the white matter, which is responsible for communicationbetween axons. The white matter appears white because of the multiplelayers formed by the myelin sheaths around the axons, which are theorigin of the high, inhomogeneous and anisotropic scattering propertiesof brain, and is a suitable surrogate for use in neural tissue opticscalculations with published optical properties, such as those below forfeline white matter.

λ ∝_(∝) ∝_(σ) [νμ] ∝_(σ) [χμ⁻¹] [χμ⁻¹] [χμ⁻¹] ∝_(εφφ) [χμ⁻¹] δ [χμ] 63352.6 1.58 .80 10.52 7.5 0.14 514 — — — 10.9 0.091 488 — — — 13.3 0.075

As was described earlier, the one-dimensional irradiance profile intissue, I, obeys the following relation, I=I_(o)e^(−(Qμz)), where Q isthe volume fraction of the characterized material that is surrounded byan optically neutral substance such as interstitial fluid or physiologicsaline. In the case of most nerves, Q=0.45 can be estimated fromcross-sectional images. The optical transport properties of tissue yieldan exponential decrease of the irradiance (ignoring temporal spreading,which is inconsequential for this application) through the target, orthe tissue surrounding the target(s). The plot above contains goodagreement between theory and model, validating the approach. It can bealso seen that the optical penetration depth, as calculated by the aboveoptical parameters agrees reasonably well with the experimentalobservations of measured response vs. irradiance for the exampledescribed above.

Furthermore, the use of multidirectional illumination, as has beendescribed herein, may serve to reduce this demand, and thus the targetradius may be considered as the limiting geometry, and not the diameter.For instance, if the abovementioned case of illuminating a 1 mm nervefrom 2 opposing sides instead of just the one, we can see that we willonly need an irradiance of ˜6 mW/mm² because the effective thickness ofthe target tissue is now ½ of what it was. It should be noted that thisis not a simple linear system, or the irradiance value would have been20/2=10 mW/mm². The discrepancy lies in the exponential nature of thephoton transport process, which yields the severe diminution of theincident power at the extremes of the irradiation field. Thus, there isa practical limit to the number of illuminations directions that providean efficiency advantage for deep, thick, and/or embedded tissue targets.

By way of non-limiting example, a 2 mm diameter nerve target may beconsidered a 1 mm thick target when illuminated circumferentially.Values of the sizes of a few key nerves follows as a set of non-limitingexamples. The diameter of the main trunk of the pudendal nerve is4.67±1.17 mm, whereas the branches of the ulnar nerve range in diameterfrom about 0.7-2.2 mm and the vagus nerve in the neck between 1.5-2.5mm.

Circumferential, and/or broad illumination may be employed to achieveelectrically and optically efficient optogenetic target activation forlarger structures and/or enclosed targets that cannot be addresseddirectly. This is illustrated in FIG. 8, where Optical Fibers OF1 andOF2 now illuminate the targeted tissue structure (N) from diametricallyopposing sides with Illumination Fields I1 and I2, respectively.Alternately, the physical length of the illumination may be extended toprovide for more photoactivation of expressed opsin proteins, withoutthe commensurate heat build up associated with intense illuminationlimited to smaller area. That is, the energy may be spread out over alarger area to reduce localized temperature rises. In a furtherembodiment, the applicator may contain a temperature sensor, such as aresistance temperature detector (RTD), thermocouple, or thermistor, etc.to provide feedback to the processor in the housing to assure thattemperature rises are not excessive, as is discussed in further detailbelow.

From the examples above, activation of a neuron, or set(s) of neuronswithin a 2.5 mm diameter vagus nerve may be nominally circumferentiallyilluminated by means of the optical applicators described later using anexternal surface irradiance of ≥5.3 mW/mm², as can be seen using theabove curve when considering the radius as the target tissue thickness,as before. However, this is greatly improved over the 28 mW/mm² requiredfor a 2.5 mm target diameter, or thickness. In this case, 2 sets of theopposing illumination systems from the embodiment above may be used, asthe target surface area has increased, configuring the system to useOptical Fibers OF3 and OF4 to provide Illumination Fields I3 and I4, asshown in FIG. 9. There are also thermal concerns to be understood andaccounted for in the design of optogenetic systems, and excessiveirradiances will cause proportionately large temperature rises. Thus, itmay be beneficial to provide more direct optical access to targetsembedded in tissues with effective depths of greater than ˜2 mm becauseof the regulatory limit applied to temperature rise allowed byconventional electrical stimulation, or “e-stim”, devices of ΔT≤2.0° C.

As described above, optical applicators suitable for use with thepresent invention may be configured in a variety of ways. Referring toFIGS. 10A-10C, a helical applicator with a spring-like geometry isdepicted. Such a configuration may be configured to readily bend with,and/or conform to, a targeted tissue structure (N), such as a nerve,nerve bundle, vessel, or other structure to which it is temporarily orpermanently coupled. Such a configuration may be coupled to suchtargeted tissue structure (N) by “screwing” the structure onto thetarget, or onto one or more tissue structures which surround or arecoupled to the target. As shown in the embodiment of FIG. 10A, awaveguide may be connected to, or be a contiguous part of, a deliverysegment (DS), and separable from the applicator (A) in that it may beconnected to the applicator via connector (C). Alternately, it may beaffixed to the applicator portion without a connector and not removable.Both of these embodiments are also described with respect to thesurgical procedure described herein. Connector (C) may be configured toserve as a slip-fit sleeve into which both the distal end of DeliverySegment (DS) and the proximal end of the applicator are inserted. In thecase where the delivery segment is an optical conduit, such an opticalfiber, it preferably should be somewhat undersized in comparison to theapplicator waveguide to allow for axial misalignment. For example, a 50μm core diameter fiber may be used as delivery segment (DS) to couple toa 100 μm diameter waveguide in the applicator (A). Such 50 μm axialtolerances are well within the capability of modern manufacturingpractices, including both machining and molding processes. The termwaveguide is used herein to describe an optical conduit that confineslight to propagate nominally within it, albeit with exceptions foroutput coupling of the light, especially to illuminate the target.

FIG. 64 shows an exemplary embodiment, wherein Connector C may comprisea single flexible component made of a polymer material to allow it tofit snugly over the substantially round cross-sectional Delivery SegmentDS1, and Applicator A. These may be waveguides such as optical fibersand similar mating structures on the applicator, and/or deliverysegment, and/or housing to create a substantially water-tight seal,shown as SEAL1 & SEAL2, that substantially prevents cells, tissues,fluids, and/or other biological materials from entering the OpticalInterface O-INT.

FIG. 65 shows an alternate exemplary embodiment, wherein Connector C maycomprise a set of seals, shown as SEAL0 through SEAL4, rather than relyupon the entire device to seal the optical connection. A variety ofdifferent sealing mechanisms may be utilized, such as, by way ofnon-limiting example, o-rings, single and dual lip seals, and wiperseals. The materials that may be used, by way of non-limiting example,are Nitrile (NBR, such as S1037), Viton, Silicone (VMQ, such as V1039,S1083 and S1146), Neoprene, Chloroprene (CR), Ethylene Propylene (EPDM,such as E1074 and E1080), Polyacrylic (ACM), Styrene Butadiene Rubber(SBR), and Fluorosilicone (FVMQ). SEAL0 through SEAL4 are shown in theexemplary embodiment to be resident within a Seal Bushing SB.

Alternately, the seal may be a component of the delivery segment and/orthe housing, and/or the applicator, thus eliminating one insertion sealwith a fixed seal, which may improve the robustness of the system. Sucha hybrid system is shown in FIG. 66, where SEAL1 is shown as an integralseal permanently linking Applicator A with its subcomponent Connector Csuch that the connection at Optical Interface O-INT is established byinserting Delivery Segment DS1 into Connector C, and having seals SEAL2,SEAL3, and SEAL4 create the substantially water-tight seal aboutDelivery Segment DS1, while SEAL1 is integrated into Connector C.

Alternately, or in addition to the other embodiments, a biocompatibleadhesive, such as, by way of non-limiting example, Loctite 4601, may beused to adhere the components being connected. Although other adhesivesare considered within the scope of the present invention, cyanoacrylatessuch as Loctite 4601, have relatively low shear strength, and may beovercome by stretching and separating the flexible sleeve from the matedcomponents for replacement without undue risk of patient harm. However,care must be taken to maintain clarity at Optical Interface O-INT.

FIG. 67 shows an alternate exemplary embodiment, wherein Connector C mayfurther comprise a high precision sleeve, Split Sleeve SSL, which isconfigured to axially align the optical elements at Optical InterfaceO-INT. By way of non-limiting example, split zirconia ceramic sleevesfor coupling both Ø1.25 and Ø2.5 mm fiber optic ferrules, not shown, maybe used to provide precision centration and all those components areavailable from Adamant-Kogyo. Similarly, other diameters may beaccommodated using the same split sleeve approach to butt-couplingoptical elements, such as optical fibers themselves.

FIG. 68 shows an alternate exemplary embodiment, wherein the seals ofFIGS. 66-67 of Connector C have been replaced by an integral sealingmechanism comprised of seals SEAL2 through SEAL4, that serve to fitabout the circumference of Delivery Segment DS1, and create gaps GAP1and GAP2. Rather than utilizing separate sealing elements, the sealingelements as shown are made to be part of an integrated sleeve.

Alternately, although not shown, the sealing mechanism may be configuredto utilize a threaded mechanism to apply axial pressure to the sealingelements to create a substantially water-tight seal that substantiallyprevents cells, tissues, fluids, and/or other biological materials fromentering the optical interfaces.

As shown in FIGS. 10 and 64-68, the optical elements being connected byConnector C may be optical fibers, as shown in the exemplaryembodiments. They may also be other portions of the therapeutic system,such as the delivery segments, an optical output from the housing, andan applicator itself.

Biocompatible adhesive may be applied to the ends of connector (C) toensure the integrity of the coupling. Alternately, connector (C) may beconfigured to be a contiguous part of either the applicator or thedelivery device. Connector (C) may also provide a hermetic electricalconnection in the case where the light source is located at theapplicator. In this case, it may also serve to house the light source,too. The light source may be made to butt-couple to the waveguide of theapplicator for efficient optical transport. Connector (C) may becontiguous with the delivery segment or the applicator. Connector (C)may be made to have cross-sectional shape with multiple internal lobessuch that it may better serve to center the delivery segment to theapplicator.

The applicator (A) in this embodiment also comprises a Proximal Junction(PJ) that defines the beginning of the applicator segment that is inoptical proximity to the target nerve. That is, PJ is the proximallocation on the applicator optical conduit (with respect to thedirection the light travels into the applicator) that is well positionedand suited to provide for light output onto the target. The segment justbefore PJ is curved, in this example, to provide for a more linearaspect to the overall device, such as might be required when theapplicator is deployed along a nerve, and is not necessarily well suitedfor target illumination. Furthermore, the applicator of this exemplaryembodiment also comprises a Distal Junction (DJ), and Inner Surface(IS), and an Outer Surface (OS). Distal Junction (DJ) represents thefinal location of the applicator still well positioned and suited toilluminate the target tissue(s). However, the applicator may extendbeyond DJ, no illumination is intended beyond DJ. DJ may also be made tobe a reflective element, such as a mirror, retro-reflector, diffusereflector, a diffraction grating, A Fiber Bragg Grating (“FBG”—furtherdescribed below in reference to FIG. 12), or any combination thereof. Anintegrating sphere made from an encapsulated “bleb” of BaSO₄, or othersuch inert, non-chromophoric compound may serve a diffuse reflector whenpositioned, for example, at the distal and of the applicator waveguide.Such a scattering element should also be placed away from the targetarea, unless light that is disallowed from waveguiding due to itsspatial and/or angular distribution is desired for therapeuticillumination.

Inner Surface (IS) describes the portion of the applicator that “faces”the target tissue, shown here as Nerve (N). That is, N lies within thecoils of the applicator and is in optical communication with IS. Thatis, light exiting IS is directed towards N. Similarly, Outer Surface(OS) describes that portion of the applicator that is not in opticalcommunication with the target. That is, the portion that faces outwards,away from the target, such a nerve that lies within the helix. OuterSurface (OS) may be made to be a reflective surface, and as such willserve to confine the light within the waveguide and allow for output tothe target via Inner Surface (IS). The reflectivity of OS may beachieved by use of a metallic or dielectric reflector deposited alongit, or simply via the intrinsic mechanism underlying fiber optics, totalinternal reflection (“TIR”). Furthermore, Inner Surface (IS) may beconditioned, or affected, such that it provides for output coupling ofthe light confined within the helical waveguide. The term outputcoupling is used herein to describe the process of allowing light toexit the waveguide in a controlled fashion, or desired manner. Outputcoupling may be achieved in various ways. One such approach may be totexture IS such that light being internally reflected no longerencounters a smooth TIR interface. This may be done along IScontinuously, or in steps. The former is illustrated in FIG. 11A in aschematic representation of such a textured applicator, as seen from IS.Surface texture is synonymous with surface roughness, or rugosity. It isshown in the embodiment of FIG. 11A as being isotropic, and thus lackinga definitive directionality. The degree of roughness is proportional tothe output coupling efficiency, or the amount of light removed form theapplicator in proportion to the amount of light encountering theTextured Area. In one embodiment, the configuration may be envisioned asbeing akin to what is known as a “matte finish”, whereas OS will may beconfigured to have a more planar and smooth finish, akin to what isknown as a “gloss finish”. A Textured Area may be an area along orwithin a waveguide that is more than a simple surface treatment. Itmight also comprise a depth component that either diminishes thewaveguide cross sectional area, or increases it to allow for outputcoupling of light for target illumination.

In this non-limiting example, IS contains areas textured with TexturedAreas TA correspond to output couplers (OCs), and between them areUntextured Areas (UA). Texturing of textured Areas (TA) may beaccomplished by, for example, mechanical means (such as abrasion) orchemical means (such as etching). In the case where optical fiber isused as the basis for the applicator, one may first strip buffer andcladding layers which may be coupled to the core, to expose the core fortexturing.

The waveguide may lay flat (with respect to gravity) for more uniformdepth of surface etching, or may be tilted to provide for a morewedge-shaped etch.

Referring to the schematic representation of FIG. 11B, an applicator isseen from the side with IS facing downward, and TA that do not wraparound the applicator to the outer surface (OS). Indeed, in suchembodiment, they need not wrap even halfway around: because the texturemay output couple light into a broad solid angle, Textured Areas (TA)need not be of large radial angular extent.

In either case, the proportion of light coupled out to the target alsomay be controlled to be a function of the location along the applicatorto provide more uniform illumination output coupling from IS to thetarget, as shown in FIGS. 11-12 and 20-23. This may be done to accountfor the diminishing proportion of light encountering later (or distal)output coupling zones. For example, if we consider the three (3) outputcoupling zones represented by Textured Areas (TA) in the presentnon-limiting example schematically illustrated in FIG. 11B, we now haveTA1, TA2, and TA3. In order to provide equal distribution of the outputcoupled energy (or power) the output coupling efficiencies would be asfollows: TA1=33%, TA2=50%, TA3=100%. Of course, other such portioningschemes may be used for different numbers of output coupling zones TAx,or in the case where there is directionality to the output couplingefficiency and a retro-reflector is used in a two-pass configuration, asis described in further detail below.

Referring to FIG. 11C, in the depicted alternate embodiment, distaljunction (DJ) is identified to make clear the distinction of the size ofTA with respect to the direction of light propagation.

In another embodiment, as illustrated in FIG. 11D, Textured Areas TA1,TA2 and TA3 are of increasing size because they are progressively moredistal with the applicator. Likewise, Untextured Areas UA1, UA2 and UA3are shown to become progressively smaller, although they also may bemade constant. The extent (or separation, size, area, etc.) of theUntextured Areas (UAx) dictates the amount of illumination zone overlap,which is another means by which the ultimate illumination distributionmay be controlled and made to be more homogeneous in ensemble. Note thatOuter Surface (OS) may be made to be reflective, as described earlier,to prevent light scattered from a TA to escape the waveguide via OS andenhance the overall efficiency of the device. A coating may be used forthe reflective element. Such coating might be, for example, metalliccoatings, such as, Gold, Silver, Rhodium, Platinum, Aluminum.Alternately, a diffusive coating of a non-chromophoric substance, suchas, but not limited to, BaSO₄ may be used as a diffuse reflector.

In a similar manner, the surface roughness of the Textured Areas (TA)may be changed as a function of location along the applicator. Asdescribed above, the amount of output coupling is proportional to thesurface rugosity, or roughness. In particular, it is proportional to thefirst raw moment (“mean”) of the distribution characterizing the surfacerugosity. The uniformity in both it spatial and angular emission areproportional to the third and fourth standardized moments (or “skewness”and “kurtosis”), respectively. These are values that may be adjusted, ortailored, to suit the clinical and/or design need in a particularembodiment. Also, the size, extent, spacing and surface roughness mayeach be employed for controlling the amount and ensemble distribution ofthe target illumination.

Alternately, directionally specific output coupling maybe employed thatpreferentially outputs light traveling in a certain direction by virtueof the angle it makes with respect to IS. For example, a wedge-shapedgroove transverse to the waveguide axis of IS will preferentially couplelight encountering it when the angle incidence is greater than thatrequired for TIR. If not, the light will be internally reflected andcontinue to travel down the applicator waveguide.

Furthermore, in such a directionally specific output couplingconfiguration, the applicator may utilize the abovementionedretro-reflection means distal to DJ. FIG. 12 illustrates an examplecomprising a FBG retro-reflector.

A waveguide, such as a fiber, can support one or even many guided modes.Modes are the intensity distributions that are located at or immediatelyaround the fiber core, although some of the intensity may propagatewithin the fiber cladding. In addition, there is a multitude of claddingmodes, which are not restricted to the core region. The optical power incladding modes is usually lost after some moderate distance ofpropagation, but can in some cases propagate over longer distances.Outside the cladding, there is typically a protective polymer coating,which gives the fiber improved mechanical strength and protectionagainst moisture, and also determines the losses for cladding modes.Such buffer coatings may consist of acrylate, silicone or polyimide. Forlong-term implantation in a body, it may be desirable to keep moistureaway from the waveguide to prevent refractive index changes that willalter the target illumination distribution and yield other commensuratelosses. Therefore, for long-term implantation, a buffer layer (orregion) may be applied to the Textured Areas TAx of the applicatorwaveguide. In one embodiment, “long-term” may be defined as greater thanor equal to 2 years. The predominant deleterious effect of moistureabsorption on optical waveguides is the creation of hydroxyl absorptionbands that cause transmission losses in the system. This is a negligiblefor the visible spectrum, but an issue for light with wavelengths longerthan about 850 nm. Secondarily, moisture absorption may reduce thematerial strength of the waveguide itself and lead to fatigue failure.Thus, while moisture absorption is a concern, in certain embodiments itis more of a concern for the delivery segments, which are more likely toundergo more motion and cycles of motion than the applicator.

Furthermore, the applicator maybe enveloped or partially enclosed by ajacket, such as Sleeve S shown in FIG. 10B. Sleeve S may be made to be areflector, as well, and serve to confine light to the intended target.Reflective material(s), such as Mylar, metal foils, or sheets ofmultilayer dielectric thin films may be located within the bulk ofSleeve S, or along its inner or outer surfaces. While the outer surfaceof Sleeve S also may be utilized for reflective purposes, in certainembodiments such a configuration is not preferred, as it is in moreintimate contact with the surrounding tissue than the inner surface.Such a jacket may be fabricated from polymeric material to provide thenecessary compliance required for a tight fit around the applicator.Sleeve S, or an adjunct or alternative to, may be configured such thatits ends slightly compress the target over a slight distance, butcircumferentially to prevent axial migration, infiltration along thetarget surface. Sleeve S may also be made to be highly scattering(white, high albedo) to serve as diffusive retro-reflector to improveoverall optical efficiency by redirecting light to the target.

Fluidic compression may also be used to engage the sleeve over theapplicator and provide for a tighter fit to inhibit proliferation ofcells and tissue ingrowth that may degrade the optical delivery to thetarget. Fluidic channels may be integrated into Sleeve S and filled atthe time of implantation. A valve or pinch-off may be employed to sealthe fluidic channels. Further details are described herein.

Furthermore, Sleeve S may also be made to elute compounds that inhibitscar tissue formation. This may provide for increased longevity of theoptical irradiation parameters that might otherwise be altered by theformation of a scar, or the infiltration of tissue between theapplicator and the target. Such tissue may scatter light and diminishthe optical exposure. However, the presence of such infiltrates couldalso be detected by means of an optical sensor placed adjacent to thetarget or the applicator. Such a sensor could serve to monitor theoptical properties of the local environment for system diagnosticpurposes. Sleeve S may also be configured to utilize a joining meansthat is self-sufficient, such as is illustrated in the cross-section ofFIG. 10C, wherein at least a part of the applicator is shown enclosed incross-section A-A. Alternately, Sleeve S may be joined using sutures orsuch mechanical or geometric means of attachment, as illustrated byelement F in the simplified schematic of FIG. 10C.

In a further embodiment, output coupling may be achieved by means oflocalized strain-induced effects with the applicator waveguide thatserve to alter the trajectory of the light within it, or the bulkrefractive index on the waveguide material itself, such as the use ofpolarization or modal dispersion. For example, output coupling may beachieved by placing regions (or areas, or volumes) of form-inducedrefractive index variation and/or birefringence that serve to alter thetrajectory of the light within the waveguide beyond the critical anglerequired for spatial confinement and/or by altering the value of thecritical angle, which is refractive-index-dependent. Alternately, theshape of the waveguide may be altered to output couple light from thewaveguide because the angle of incidence at the periphery of thewaveguide has been modified to be greater than that of the criticalangle required for waveguide confinement. These modifications may beaccomplished by transiently heating, and/or twisting, and/or pinchingthe applicator in those regions where output coupling for targetillumination is desired. A non-limiting example is shown in FIG. 14,where a truncated section of Waveguide WG has been modified betweenEndpoints (EP) and Centerpoint (CP). The cross-sectional area and/ordiameter of CP<EP. Light propagating through Waveguide WG will encountera higher angle of incidence at the periphery of the waveguide due to themechanical alteration of the waveguide material, resulting in lightoutput coupling near CP in this exemplary configuration. It should benoted that light impinging upon the relatively slanted surface providedby the taper between EP and CP may output couple directly from the WGwhen the angle is sufficiently steep, and may require more than a singleinteraction with said taper before its direction is altered to such adegree that is ejected from the WG. As such, consideration may be givento which side of the WG is tapered, if it is not tapered uniformly, suchthat the output coupled light exiting the waveguide is directed towardthe target, or incident upon an alternate structure, such as a reflectorto redirect it to the target.

Referring to FIG. 13 and the description that follows, for contextualpurposes an exemplary scenario is described wherein a light ray isincident from a medium of refractive index “n” upon a core of index“n_(core)” at a maximum acceptance angle, θ_(max), with Snell's law atthe medium-core interface being applied. From the geometry illustratedin FIG. 13, we have:

From the geometry of the above figure we have:

sin  θ_(r) = sin (90^(∘) − θ_(c)) = cos  θ_(c) where$\theta_{c} = {\sin^{- 1}\frac{n_{clad}}{n_{core}}}$

is the critical angle for total internal reflection.

Substituting cos θc for sin θr in Snell's law we get:

${\frac{n}{n_{core}}\sin \; \theta_{\max}} = {\cos \; {\theta_{c}.}}$

By squaring both sides we get:

${\frac{n^{2}}{n_{core}^{2}}\sin^{2}\; \theta_{\max}} = {{\cos^{2}\; \theta_{c}} = {{1 - {\sin^{2}\theta_{c}}} = {1 - {\frac{n_{clad}^{2}}{n_{core}^{2}}.}}}}$

Solving, we find the formula stated above:

n sin θ_(max)=√{square root over (n _(core) ² −n _(clad) ²)},

This has the same form as the numerical aperture (NA) in other opticalsystems, so it has become common to define the NA of any type of fiberto be

NA=√{square root over (n _(core) ² −n _(clad) ²)},

It should be noted that not all of the optical energy impinging at lessthan the critical angle will be coupled out of the system.

Alternately, the refractive index may be modified using exposure toultraviolet (UV) light, such might be done to create a Fiber BraggGrating (FBG). This modification of the bulk waveguide material willcause the light propagating through the waveguide to refractive togreater or lesser extent due to the refractive index variation. Normallya germanium-doped silica fiber is used in the fabrication of suchrefractive index variations. The germanium-doped fiber isphotosensitive, which means that the refractive index of the corechanges with exposure to UV light.

Alternately, and/or in combination with the abovementioned aspects andembodiments of the present invention, “whispering gallery modes” may beutilized within the waveguide to provide for enhanced geometric and/orstrain-induced output coupling of the light along the length of thewaveguide. Such modes of propagation are more sensitive to small changesin the refractive index, birefringence and the critical confinementangle than typical waveguide-filling modes because they are concentratedabout the periphery of a waveguide. Thus, they are more susceptible tosuch means of output coupling and provide for more subtle means ofproducing a controlled illumination distribution at the target tissue.

Alternately, more than a single Delivery Segment DS may be brought fromthe housing (H) to the applicator (A), as shown in FIG. 15. HereDelivery Segments DS1 and DS2 are separate and distinct. They may carrylight from different sources (and of different color, or wavelength, orspectra) in the case where the light is created in housing (H), or theymay be separate wires (or leads, or cables) in the case where the lightis created at or near applicator (A).

In either case, the applicator may alternately further comprise separateoptical channels for the light from the different Delivery Segments DSx(where x denotes the individual number of a particular delivery segment)in order to nominally illuminate the target area. A further alternateembodiment may exploit the inherent spectral sensitivity of theretro-reflection means to provide for decreased output coupling of onechannel over another. Such would be the case when using a FBGretro-reflector, for instance. In this exemplary case, light of a singlecolor, or narrow range of colors will be acted on by the FBG. Thus, itwill retro-reflect only the light from a given source for bi-directionaloutput coupling, while light from the other source will pass throughlargely unperturbed and be ejected elsewhere. Alternately, a chirped FBGmay be used to provide for retro-reflection of a broader spectrum,allowing for more than a single narrow wavelength range to be acted uponby the FBG and be utilized in bi-directional output coupling. Of course,more than two such channels and/or Delivery Segments (DSx) are alsowithin the scope of the present invention, such as might be the casewhen selecting to control the directionality of the instigated nerveimpulse, as will be described in a subsequent section.

Alternately, multiple Delivery Segments may also provide light to asingle applicator, or become the applicator(s) themselves, as isdescribed in further detail below. For example, a single optical fiberdeployed to the targeted tissue structure, wherein the illumination isachieved through the end face of the fiber is such a configuration,albeit a simple one. In this configuration, the end face of the fiber isthe output coupler, or, equivalently, the emission facet, as the termsare interchangeable as described herein.

Alternately, a single delivery device may used to channel light frommultiple light sources to the applicator. This may be achieved throughthe use of spliced, or conjoined, waveguides (such as optical fibers),or by means of a fiber switcher, or a beam combiner prior to initialinjection into the waveguide, as shown in FIG. 16.

In this embodiment, Light Sources LS1 and LS2 output light along pathsW1 and W2, respectively. Lenses L1 and L2 may be used to redirect thelight toward Beam Combiner (BC), which may serve to reflect the outputof one light source, while transmitting the other. The output of LS1 andLS2 may be of different color, or wavelength, or spectral band, or theymay be the same. If they are different, BC may be a dichroic mirror, orother such spectrally discriminating optical element. If the outputs ofLight Sources LS1 and LS2 are spectrally similar, BC may utilizepolarization to combine the beams. Lens L3 may be used to couple the W1and W2 into Waveguide (WG). Lenses L1 and L2 may also be replaced byother optical elements, such as mirrors, etc. This method is extensibleto greater numbers of light sources.

The type of optical fiber that may be used as either delivery segmentsor within the applicators is varied, and may be selected from the groupconsisting of: Step-index, GRIN (“gradient index”), Power-Law index,etc. Alternately, hollow-core waveguides, photonic crystal fiber (PCF),and/or fluid filled channels may also be used as optical conduits. PCFis meant to encompass any waveguide with the ability to confine light inhollow cores or with confinement characteristics not possible inconventional optical fiber. More specific categories of PCF includephotonic-bandgap fiber (PBG, PCFs that confine light by band gapeffects), holey fiber (PCFs using air holes in their cross-sections),hole-assisted fiber (PCFs guiding light by a conventional higher-indexcore modified by the presence of air holes), and Bragg fiber (PBG formedby concentric rings of multilayer film). These are also known as“microstructured fibers”. End-caps or other enclosure means may be usedwith open, hollow waveguides such as tubes and PCF to prevent fluidinfill that would spoil the waveguide.

PCF and PBG intrinsically support higher numerical aperture (NA) thanstandard glass fibers, as do plastic and plastic-clad glass fibers.These provide for the delivery of lower brightness sources, such asLEDs, OLEDs, etc. This is notable for certain embodiments because suchlower brightness sources are typically more electrically efficient thanlaser light sources, which is relevant for implantable deviceembodiments in accordance with the present invention that utilizebattery power sources. Configurations for to creating high-NA waveguidechannels are described in greater detail herein.

Alternately, a bundle of small and/or single mode (SM) opticalfibers/waveguides may be used to transport light as delivery segments,and/or as an applicator structure, such as is shown in a non-limitingexemplary embodiment in FIG. 17A. In this embodiment, Waveguide (WG) maybe part of the Delivery Segment(s) (DS), or part of the applicator (A)itself. As shown in the embodiment of FIG. 17A, the waveguide (WG)bifurcates into a plurality of subsequent waveguides, BWGx. The terminusof each BWGx is Treatment Location (TLx). The terminus may be the areaof application/target illumination, or may alternately be affixed to anapplicator for target illumination. Such a configuration is appropriatefor implantation within a distributed body tissue, such as, by way ofnon-limiting example, the liver, pancreas, or to access cavernousarteries of the corpora cavernosa (to control the degree of smoothmuscle relaxation in erection inducement).

Referring to FIG. 17B, the waveguide (WG) may also be configured toinclude Undulations (U) in order to accommodate possible motion and/orstretching/constricting of the target tissues, or the tissuessurrounding the target tissues, and minimize the mechanical load (or“strain”) transmitted to the applicator from the delivery segment andvice versa. Undulations (U) may be pulsed straight during tissueextension and/or stretching. Alternately, Undulations (U) may beintegral to the applicators itself, or it may be a part of the DeliverySegments (DS) supplying the applicator (A). The Undulations (U) may bemade to areas of output coupling in embodiments when the Undulations (U)are in the applicator. This may be achieved by means of similarprocesses to those described earlier regarding means by which to adjustthe refractive index and/or the mechanical configuration(s) of thewaveguide for fixed output coupling in an applicator. However, in thiscase, the output coupling is achieved by means of tissue movement thatcauses such changes. Thus, output coupling is nominally only providedduring conditions of tissue extension and/or contraction and/or motion.The Undulations (U) may be configured of a succession of waves, or bendsin the waveguide, or be coils, or other such shapes. Alternately, DScontaining Undulations (U) may be enclosed in a protective sheath orjacket to allow DS to stretch and contract without encountering tissuedirectly.

A rectangular slab waveguide may be configured to be like that of theaforementioned helical-type, or it can have a permanent waveguide (WG)attached/inlaid. For example, a slab may be formed such that is alimiting case of a helical-type applicator, such as is illustrated inFIG. 18 for explanatory purposes and to make the statement that theattributes and certain details of the aforementioned helical-typeapplicators are suitable for this slab-like as well and need not berepeated.

In the embodiment depicted in FIG. 18, Applicator (A) is fed by DeliverySegment (DS) and the effectively half-pitch helix is closed along thedepicted edge (E), with closure holes (CH) provided, but not required.Of course, this is a reduction of the geometries discussed previously,and meant to convey the abstraction and interchangeability of the basicconcepts therein and between those of the slab-type waveguides to bediscussed.

It should also be understood that the helical-type applicator describedherein may also be utilized as a straight applicator, such as may beused to provide illumination along a linear structure like a nerve, etc.A straight applicator may also be configured as the helical-typeapplicators described herein, such as with a reflector to redirect straylight toward the target, as is illustrated in FIG. 19A by way ofnon-limiting example.

Here Waveguide (WG) contains Textured Area (TA), and the addition ofReflector (M) that at least partially surrounds target anatomy (N). Thisconfiguration provides for exposure of the far side of the target byredirecting purposefully exposed and scattered light toward the side ofthe target opposite the applicator. FIG. 19B illustrates the sameembodiment, along cross-section A-A, showing schematically the use of amirror (as Reflector M) surrounding Target (N.) Although not shown, WGand M may be affixed to a common casing (not shown) that forms part ofthe applicator. Reflector (M) is shown as being comprised of a pluralityof linear faces, but need not be. In one embodiment it may be made to bea smooth curve, or in another embodiment, a combination of the two.

In another alternate embodiment, a straight illuminator may be affixedto the target, or tissue surrounding or adjacent or nearby to the targetby means of the same helix-type (“helical”) applicator. However, in thiscase the helical portion is not the illuminator, it is the means toposition and maintain another illuminator in place with respect to thetarget. The embodiment illustrated in FIG. 20 utilizes thetarget-engaging feature(s) of the helical-type applicator to locatestraight-type Applicator (A) in position near Target (N) via ConnectorElements CE1 and CE2, which engage the Support Structure (D) to locateand maintain optical output. Output illumination is shown as beingemitted via Textured Area (TA), although, as already discussed,alternate output coupling means are also within the scope of the presentinvention. The generality of the approach and the interchangeability ofthe different target-engaging means described herein (even subsequent tothis section) are also applicable to serve as such Support Structures(D), and therefore the combination of them is also within the scope ofthe present invention.

Slab-type (“slab-like”) geometries of Applicator A, such as thin, planarstructures, can be implanted, or installed at, near, or around thetissue target or tissue(s) containing the intended target(s). Anembodiment of such a slab-type applicator configuration is illustratedin FIGS. 21A-21C. It may be deployed near or adjacent to a targettissue, and it may also be rolled around the target tissue, or tissuessurrounding the target(s). It may be rolled axially, as illustrated byelement AM1 in FIG. 21B, (i.e. concentric with the long axis of thetargeted tissue structure N), or longitudinally, as illustrated byelement AM2 in FIG. 21C (i.e. along the long axis of target N), asrequired by the immediate surgical situation, as shown in the moredetailed figure below. The lateral edges that come into contact witheach other once deployed at the target location could be made withcomplementary features to assure complete coverage and limit the amountof cellular infiltrate (i.e. limit scar tissue or other opticalperturbations over time to better assure an invariant target irradiance,as was described in the earlier section pertaining to the helical-typeapplicator). Closure Holes (CH) are provided for this purpose in thefigure of this non-limiting example. The closure holes (CH) may besutured together, of otherwise coupled using a clamping mechanism (notshown). It may also provide different output coupling mechanisms thanthe specific helical-type waveguides described above, although, it is tobe understood that such mechanisms are fungible, and may be usedgenerically. And vice versa, that elements of output coupling, opticalrecirculation and waveguiding structures, as well as deploymenttechniques discussed in the slab-type section maybe applicable tohelical-type, and straight waveguides.

The slab-type applicator (A) illustrated in FIGS. 21A-21C is comprisedof various components, as follows. In the order “seen” by light enteringthe applicator, first is an interface with the waveguide of the deliverysegment (DS). Alternately, the waveguide may be replaced by electricalwires, in the case where the emitter(s) is(are) included near or withinthe applicator. An Optical Plenum (OP) structure may be present afterthe interface to segment and direct light propagation to differentchannels CH using distribution facets (DF), whether it comes from thedelivery segments (DS), or from a local light source. The optical plenum(OP) may also be configured to redirect all of the light entering thelight entering it, such as might be desirable when the delivery segment(DS) should lie predominantly along the same direction as the applicator(A). Alternately, it may be made to predominantly redirect the light atangle to provide for the applicator to be directed differently than thedelivery segment(s) (DS). Light propagating along the channel(s) (CH)may encounter an output coupling means, such as Partial Output Coupler(POC) and Total Output Coupler (TOC). The proximal output couplers (POC)redirect only part of the channeled light, letting enough light pass toprovide adequate illumination to more distal targets, as was discussedpreviously. The final, or distal-most, output coupler (TOC) may be madeto redirect nominally all of the impinging light to the target. Thepresent embodiment also contains provisions for outer surface reflectorsto redirect errant light to the target. It is also configured to supporta reflector (RE) on or near the inner surface (IS) of applicator (A),with apertures (AP) to allow for the output coupled light to escape,that serves to more readily redirect any errant or scattered light backtoward the target (N). Alternately, such a reflector (RE) may beconstructed such that it is not covering the output coupler area, butproximal to it in the case of longitudinally rolled deployment such thatit nominally covers the intended target engagement area (TEA). Reflector(RE) may be made from biocompatible materials such as platinum, or goldif they are disposed along the outside of the applicator (A).Alternately, such metallic coatings may be functionalized in order tomake them bioinert, as is discussed below. The output couplers POC andTOC are shown in FIG. 21A as being located in the area of the applicator(A) suitable for longitudinal curling about the target (N) (FIG. 21B),or tissues surrounding the target (N), but need not be, as would be thecase for deployments utilizing the unrolled and axially rolledembodiments (AM1). Any such surface (or sub-surface) reflector (RE)should be present along (or throughout) a length sufficient to provideat least complete circumferential coverage once the applicator isdeployed. As used herein the terms optical conduit and channel memberare equivalent.

The current embodiment utilizes PDMS, described below, or some othersuch well-qualified polymer, as a substrate (SUB) that forms the body ofthe applicator (A), for example as in FIG. 21A. For example, biologicalmaterials such as hyaluronan, elastin, and collagen, which arecomponents of the native extracellular matrix, may also be used alone orin combination with inorganic compounds to form the substrate (SUB).Hydrogel may also be used, as it is biocompatible, may be made to elutebiological and/or pharmaceutical compounds, and has a low elasticmodulus, making it a compliant material. Likewise polyethylene, and/orpolypropylene may also be used to fro Substrate SUB.

A material with a refractive index lower than that of the substrate(SUB) (PDMS in this non-limiting example) may used as filling (LFA) tocreate waveguide cladding where the PDMS itself acts as the waveguidecore. In the visible spectrum, the refractive index of PDMS is ˜1.4.Water, and even PBS and saline have indices of ˜1.33, making themsuitable for cladding materials. They are also biocompatible and safefor use in an illumination management system as presented herein, evenif the integrity of the applicator (A) is compromised and they arereleased into the body.

Alternately, a higher index filling may be used as the waveguidechannel. This may be thought of as the inverse of the previouslydescribed geometry, where in lieu of the polymer comprising substrate(SUB), you have a liquid filling (LFA) acting as the waveguide coremedium, and the substrate (SUB) material acting as the cladding. Manyoils have refractive indices of ˜1.5 or higher, making them suitable forcore materials.

Alternately, a second polymer of differing refractive index may be usedinstead of the aforementioned liquid fillings. A high-refractive-indexpolymer (HRIP) is a polymer that has a refractive index greater than1.50. The refractive index is related to the molar refractivity,structure and weight of the monomer. In general, high molar refractivityand low molar volumes increase the refractive index of the polymer.Sulfur-containing substituents including linear thioether and sulfone,cyclic thiophene, thiadiazole and thianthrene are the most commonly usedgroups for increasing refractive index of a polymer in forming a HRIP.Polymers with sulfur-rich thianthrene and tetrathiaanthrene moietiesexhibit n values above 1.72, depending on the degree of molecularpacking. Such materials may be suitable for use as waveguide channelswithin a lower refractive polymeric substrate. Phosphorus-containinggroups, such as phosphonates and phosphazenes, often exhibit high molarrefractivity and optical transmittance in the visible light region.Polyphosphonates have high refractive indices due to the phosphorusmoiety even if they have chemical structures analogous topolycarbonates. In addition, polyphosphonates exhibit good thermalstability and optical transparency; they are also suitable for castinginto plastic lenses. Organometallic components also result in HRIPs withgood film forming ability and relatively low optical dispersion.Polyferrocenylsilanes and polyferrocenes containing phosphorus spacersand phenyl side chains show unusually high n values (n=1.74 and n=1.72),as well, and are also candidates for waveguides.

Hybrid techniques which combine an organic polymer matrix with highlyrefractive inorganic nanoparticles may be employed to produce polymerswith high n values. As such, PDMS may also be used to fabricate thewaveguide channels that may be integrated to a PDMS substrate, wherenative PDMS is used as the waveguide cladding. The factors affecting therefractive index of a HRIP nanocomposite include the characteristics ofthe polymer matrix, nanoparticles, and the hybrid technology betweeninorganic and organic components. Linking inorganic and organic phasesis also achieved using covalent bonds. One such example of hybridtechnology is the use of special bifunctional molecules, such as3-Methacryloxypropyltrimethoxysilane (MEMO), which possess apolymerisable group as well as alkoxy groups. Such compounds arecommercially available and can be used to obtain homogeneous hybridmaterials with covalent links, either by simultaneous or subsequentpolymerization reactions.

The following relation estimates the refractive index of ananocomposite,

n _(comp)=ϕ_(p) n _(p)+ϕ_(org) n _(org)

where, n_(comp), n_(p) and n_(org) stand for the refractive indices ofthe nanocomposite, nanoparticle and organic matrix,

respectively, while ϕ_(p) and ϕ_(org) represent the volume fractions ofthe nanoparticles and organic matrix, respectively.

The nanoparticle load is also important in designing HRIP nanocompositesfor optical applications, because excessive concentrations increase theoptical loss and decrease the processability of the nanocomposites. Thechoice of nanoparticles is often influenced by their size and surfacecharacteristics. In order to increase optical transparency and reduceRayleigh scattering of the nanocomposite, the diameter of thenanoparticle should be below 25 nm. Direct mixing of nanoparticles withthe polymer matrix often results in the undesirable aggregation ofnanoparticles—this may be avoided by modifying their surface, orthinning the viscosity of the liquid polymer with a solvent such asxylene; which may later be removed by vacuum during ultrasonic mixing ofthe composite prior to curing. Nanoparticles for HRIPs may be chosenfrom the group consisting of: TiO₂ (anatase, n=2.45; rutile, n=2.70),ZrO₂ (n=2.10), amorphous silicon (n=4.23), PbS (n=4.20) and ZnS(n=2.36). Further materials are given in the table below. The resultingnanocomposites may exhibit a tunable refractive index range, per theabove relation.

Substance n (413.3 nm) n (619.9 nm) Os 4.05 3.98 W 3.35 3.60 Sicrystalline 5.22 3.91 Si amorphous 4.38 4.23 Ge 4.08 5.59-5.64 GaP 4.083.33 GaAs 4.51 3.88 InP 4.40 3.55 InAs 3.20 4.00 InSb 3.37 4.19 PbS 3.884.29 PbSe 1.25-3.00 3.65-3.90 PbTe 1.0-1.8 6.40 Ag 0.17 0.13 Au 1.640.19 Cu 1.18 0.27

In one exemplary embodiment, a HRIP preparation based on PDMS and PbS,the volume fraction of particles needs to be around 0.2 or higher toyield n_(comp)≥1.96, which corresponds to a weight fraction of at least0.8 (using the density of PbS of 7.50 g cm⁻³ and of PDMS of 1.35 gcm⁻³). Such a HRIP can support a high numerical aperture (NA), which isuseful when coupling light from relatively low brightness sources suchas LEDs. The information given above allows for the recipe of otheralternate formulations to be readily ascertained.

There are many synthesis strategies for nanocomposites. Most of them canbe grouped into three different types. The preparation methods are allbased on liquid particle dispersions, but differ in the type of thecontinuous phase. In melt processing particles are dispersed into apolymer melt and nanocomposites are obtained by extrusion. Castingmethods use a polymer solution as dispersant and solvent evaporationyields the composite materials, as described earlier. Particledispersions in monomers and subsequent polymerization result innanocomposites in the so-called in situ polymerization route.

In a similar way, low refractive index composite materials have may alsobe prepared. As suitable filler materials, metals with low refractiveindices below 1, such as gold (shown in the table above) may be chosen,and the resulting low index material used as the waveguide cladding.

There are a variety of optical plenum configurations for capturing lightinput and creating multiple output channels. As shown in FIGS. 21 and 22the facets are comprised of linear faces, although other configurationsare within the scope of the invention. The angle of the face withrespect to the input direction of the light dictates the numericalaperture (NA). Alternately, curved faces may be employed for nonlinearangular distribution and intensity homogenization. A parabolic surfaceprofile may be used, for example. Furthermore, the faces need not beplanar. A three-dimensional surface may similarly be employed. Theposition of these plenum distribution facets DF may be used to dictatethe proportion of power captured as input to a channel, as well.Alternately, the plenum distribution facets DF may spatially located inaccordance with the intensity/irradiance distribution of the input lightsource. As a non-limiting example, in a configuration utilizing an inputwith a Lambertian irradiance distribution, such as that which may beoutput by an LED, the geometry of the distribution facets DF may betailored to limit the middle channel to have ⅓ of the emitted light, andthe outer channels evenly divide the remaining ⅔, such as is shown inFIG. 22 by way of non-limiting example.

Output Coupling may be achieved many ways, as discussed earlier.Furthering that discussion, and to be considered as part thereof,scattering surfaces in areas of intended emission may be utilized.Furthermore, output coupling facets, such as POC and TOC shownpreviously, may also be employed. These may include reflective,refractive, and/or scattering configurations. The height of facet may beconfigured to be in proportion to the amount or proportion of lightintercepted, while the longitudinal position dictates the outputlocation. As was also discussed previously, for systems employingmultiple serial OCs, the degree of output coupling of each may be madeto be proportional to homogenize the ensemble illumination. Asingle-sided facet within the waveguide channel may be disposed suchthat it predominantly captures light traveling one way down thewaveguide channel (or core). Alternately, a double-sided facet thatcaptures light traveling both ways down the waveguide channel (or core)to provide both forward and backward output coupling. This would be usedpredominantly with distal retroreflector designs. Such facets may beshaped as, by way of non-limiting example; a pyramid, a ramp, anupward-curved surface, a downward-curved surface, etc. FIG. 23illustrates output coupling for a ramp-shaped facet.

Light Ray ER enters (or is propagated within) Waveguide Core WG. Itimpinges upon Output Coupling Facet F and is redirected to the oppositesurface. It becomes Reflected Ray RR1, from which Output Coupled RayOCR1 is created, as is Reflected Ray RR2. OCR1 is directed at thetarget. OCR2 and RR3 are likewise created from RR2. Note that OCR2 isemitted from the same surface of WG as the facet. If there is no targetor reflector on that side, the light is lost. The depth of F is H, andthe Angle θ. Angle θ dictates the direction of RR1, and its subsequentrays. Angle α may be provided in order to allow for mold release forsimplified fabrication. It may also be used to output couple lighttraversing in the opposite direction as ER, such as might be the casewhen distal retro-reflectors are used.

Alternately, Output Coupling Facet F may protrude from the waveguide,allowing for the light to be redirected in an alternate direction, butby similar means.

The descriptions herein regarding optical elements, such as, but limitedto, Applicators and Delivery Segments may also be utilized by more thana single light source, or color of light, such as may be the case whenusing SFO, and/or SSFO opsins, as described in more detail elsewhereherein.

The waveguide channel(s) may be as described above. Use of fluidics mayalso be employed to expand (or contract) the applicator to alter themechanical fit, as was described above regarding Sleeve S. When usedwith the applicator (A), it may serve to decrease infiltratepermeability as well as to increase optical penetration viapressure-induced tissue clearing. Tissue clearing, or optical clearingas it is also known, refers to the reversible reduction of the opticalscattering by a tissue due to refractive index matching of scatterersand ground matter. This may be accomplished by impregnating tissue withsubstances (“clearing agents”) such as, x-ray contrast agents (e.g.Verografin, Trazograph, and Hypaque-60), glucose, propylene glycol,polypropylene glycol-based polymers (PPG), polyethylene glycol (PEG),PEG-based polymers, and glycerol by way of non-limiting examples. It mayalso be accomplished by mechanically compressing the tissue.

Fluidic channels incorporated into the applicator substrate may also beused to tune the output coupling facets. Small reservoirs beneath thefacets may be made to swell and in turn distend the location and/or theangle of the facet in order to adjust the amount of light and/or thedirection of that light.

Captured light may also be used to assess efficiency or functionalintegrity of the applicator and/or system by providing informationregarding the optical transport efficiency of the device/tissue states.The detection of increased light scattering may be indicative of changesin the optical quality or character of the tissue and or the device.Such changes may be evidenced by the alteration of the amount ofdetected light collected by the sensor. It may take the form of anincrease or a decrease in the signal strength, depending upon therelative positions of the sensor and emitter(s). An opposing opticalsensor may be employed to more directly sample the output, as isillustrated in FIG. 24. In this non-limiting embodiment, Light Field LFis intended to illuminate the Target (N) via output coupling from awaveguide within Applicator A, and stray light is collected by SensorSEN1. SEN1 may be electrically connected to the Housing (not shown) viaWires SW1 to supply the Controller with information regarding theintensity of the detected light. A second Sensor SEN2 is also depicted.Sensor SEN2 may be used to sample light within a (or multiple)waveguides of Applicator A, and its information conveyed to a controller(or processor) via Wires SW2. This provides additional informationregarding the amount of light propagating within the Waveguide(s) of theApplicator. This additional information may be used to better estimatethe optical quality of the target exposure by means of providing abaseline indicative of the amount of light energy or power that is beingemitted via the resident output coupler(s), as being proportional to theconducted light within the Waveguide(s).

Alternately, the temporal character of the detected signals may be usedfor diagnostic purposes. For example, slower changes may indicate tissuechanges or device aging, while faster changes could be strain, ortemperature dependent fluctuations. Furthermore, this signal may be usedfor closed loop control by adjusting power output over time to assuremore constant exposure at the target. The detected signal of a Sensorsuch as SEN1 may also be used to ascertain the amount of optogeneticprotein matter present in the target. If such detection is difficult tothe proportionately small effects on the signal, a heterodyned detectionscheme may be employed for this purpose. Such an exposure may be ofinsufficient duration or intensity to cause a therapeutic effect, butmade solely for the purposes of overall system diagnostics.

Alternately, an applicator may be fabricated with individuallyaddressable optical source elements to enable adjustment of theintensity and location of the light delivery, as is shown in theembodiment of FIG. 25 (1010). Such applicators may be configured todeliver light of a single wavelength to activate or inhibit nerves.Alternately, they may be configured to deliver light of two or moredifferent wavelengths, or output spectra, to provide for both activationand inhibition in a single device, or a plurality of devices.

An alternate example of such an applicator is shown in FIG. 26, whereApplicator A is comprised of Optical Source Elements LSx, may becomprised of Emitters (EM), mounted on Bases B; element “DS”xxrepresents the pertinent delivery segments as per their coordinates inrows/columns on the applicator (A); element “SUB” represents thesubstrate, element “CH” represents closure holes, and element “TA” atextured area, as described above.

The optical sensors described herein are also known as photodetectors,and come in different forms. These may include, by way of non-limitingexamples, photovoltaic cells, photodiodes, pyroelectrics,photoresistors, photoconductors, phototranisistors, and photogalvanicdevices. A photogalvanic sensor (also known as a photoelectrochemicalsensor) may be constructed by allowing a conductor, such as stainlesssteel or platinum wire, to be exposed on, at, or adjacent to a targettissue. Light being remitted from the target tissue that impinges uponthe conductor will cause it undergo a photogalvanic reaction thatproduces a electromotive force, or “EMF”, with respect to anotherconductor, or conductive element, that is at least substantially in thesame electrical circuit as the sensor conductor, such as it may be ifimmersed in the same electrolytic solution (such as is found within thebody). The EMF constitutes the detector response signal. That signal maythen be used as input to a system controller in order to adjust theoutput of the light source to accommodate the change. For example, theoutput of the light source may be increased, if the sensor signaldecreases and vice versa.

In an alternate embodiment, an additional sensor, SEN2, may also beemployed to register signals other than those of sensor SEN1 for thepurposes of further diagnosing possible causes of systemic changes.

For example, the target opacity and/or absorbance may be increasing ifSEN2 maintains a constant level indicating that the optical powerentering the applicator is constant, but sensor SEN1 shows a decreasinglevel. A commensurate decrease in the response of sensor SEN2 wouldindicate that the electrical power to the light source should beincreased to accommodate a decline in output and/or efficiency, as mightbe experienced in an aging device. Thus, an increase in optical powerand/or pulse repetition rate delivered to the applicator may mitigatethe risk of underexposure to maintain a therapeutic level.

Changes to the optical output of the light source may be made to, forexample, the output power, exposure duration, exposure interval, dutycycle, pulsing scheme

For the exemplary configuration shown in FIG. 24, the following tablemay be used to describe exemplary programming for the controller in eachcase of sensor response changes.

SEN1 SEN2 Response Response Possible Change Change Possible Cause (s)Action (s) Decrease Decrease Light source Increase output or overallelectrical optical system input power to efficiency light source todiminishing. increase optical output power and regain expected signalfrom SEN1 and/or SEN2, and/or monitor therapeutic outcome. Otherwise,replacement of the light source is possibly indicated. Decrease ConstantChange in target Increase optical electrical characteristics, inputpower to such as tissue or light source to cellular ingrowth increasebetween the optical output applicator and power and target tissue, orregain expected relative movement signal from between SEN1 whileapplicator and resetting target. baseline for SEN2 signal level, and/ormonitor therapeutic outcome. Otherwise, replacement of the applicator ispossibly indicated. Decrease Increase The amount of Increase lightdiverted to electrical SEN2 increasing. input power to light source toincrease optical output power and regain expected signal from SEN1 whileresetting baseline for SEN2 signal level, and/or monitor therapeuticoutcome. Otherwise, replacement of the applicator is possibly indicated.Constant Decrease Change in target Maintain light optical source outputcharacteristics, level while such as tissue or resetting cellularingrowth baseline for between the SEN2 signal applicator and level,and/or target tissue. monitor therapeutic outcome. Constant IncreaseChange in the Maintain light optical delivery source output efficiencyof the level while applicator. resetting baseline for SEN2 signal level.Increase Decrease Change in target Maintain light optical source outputcharacteristics, level while or movement of resetting applicator withbaseline for respect to target SEN1 and SEN2 tissue. signal levels,and/or monitor therapeutic outcome. Increase Constant Change in targetMaintain light optical source output characteristics, level while ormovement of resetting applicator with baseline for respect to targetSEN1 signal tissue. level, and/or monitor therapeutic outcome. IncreaseIncrease Change in the Decrease optical output electrical and/ordelivery input power to efficiency of the light source to system.increase optical output power and regain expected signal from SEN1 whileresetting baseline for SEN2 signal level, if original setting is notachieved, and/or monitor therapeutic outcome. Otherwise, replacement ofthe applicator is possibly indicated.

It is to be understood that the term “constant” does not simply implythat there is no change in the signal or its level, but maintaining itslevel within an allowed tolerance. Such a tolerance may be of the orderof ±20% on average. However, patient and other idiosyncrasies may alsobe need to be accounted and the tolerance band adjusted on a per patientbasis where a primary and/or secondary therapeutic outcome and/or effectis monitored to ascertain acceptable tolerance band limits. As is shownin FIG. 6, an overexposure is not expected to cause diminished efficacy.However, the desire to conserve energy while still assuring therapeuticefficacy compels that overexposures be avoided to increase both batterylifetime and the recharge interval for improved patient safety andcomfort.

Alternately, SEN2 may be what we will refer to as a therapeutic sensorconfigured to monitor a physical therapeutic outcome directly, orindirectly. Such a therapeutic sensor may be, by way of non-limitingexample, an ENG probe, an EMG probe, a pressure transducer, a chemicalsensor, an EKG sensor, or a motion sensor. A direct sensor is consideredto be one that monitors a therapeutic outcome directly, such as theaforementioned examples of chemical and pressure sensors. An indirectsensor is one that monitors an effect of the treatment, but not theultimate result. Such sensors are the aforementioned examples of ENG,EKG, and EMG probes, as are also discussed elsewhere herein.

Alternately, the therapeutic sensor may be a patient input device thatallows the patient to at least somewhat dictate the optical dosageand/or timing. Such a configuration may be utilized, by way ofnon-limiting example, in cases such as muscle spasticity, where thepatient may control the optical dosage and/or timing to provide whatthey deem to be the requisite level of control for a given situation.

In an alternate embodiment, an additional optical sensor may be locatedat the input end of the delivery segment near to the light source. Thisadditional information may assist in diagnosing system status byallowing for the optical efficiency of the delivery segments to beevaluated. For example, the delivery segments and/or their connection tothe applicator may be considered to be failing, if the output end sensorregisters a decreasing amount of light, while the input end sensor doesnot. Thus, replacing the delivery segments and/or the applicator may beindicated.

In an alternate embodiment, SEN1 may further be configured to utilize acollector, such as an optical fiber, or an t least an aspect of theApplicator itself, that serves to collect and carry the optical signalfrom, or adjacent to the Applicator to a remote location. By way ofnon-limiting example, light may be sampled at or near the target tissue,but transferred to the controller for detection and processing. Such aconfiguration is shown in FIG. 69, where Delivery Segment DS provideslight to Applicator A, creating Light Field LF. Light Field LF issampled by Collection Element COL-ELEM, which may be, by way ofnon-limiting example, a prism, a rod, a fiber, a side-firing fiber, acavity, a slab, a mirror, a diffractive element, and/or a facet.Collected Light COL-LIGHT is transmitted by Waveguide WG2 to SEN1, notshown.

Alternately, the Delivery Segment itself, or a portion thereof, may beused to transmit light to the remote location of SEN1 by means ofspectrally separating the light in the housing. This configuration maybe similar to that shown in FIG. 16, with the alterations, that LS2becomes SEN1, and Beamcombiner BC is configured such that it allowslight from the target tissue to be transmitted to SEN1, while stillallowing substantially all of the light form LS1 to be injected intoWaveguide WG for therapeutic and diagnostic purposes. Such aconfiguration may be deployed when SEN1 may be a chemometric sensor, forexample, and a fluorescence signal may be the desired measurand.

Alternate configurations are shown in FIGS. 27A (1012) and 27B (1014),wherein applicators configured as linear and planar arrays of emitters,or alternately output couplers, are shown.

A linear array optogenetic light applicator (A), which also may betermed an “optarray”, may be inserted into the intrathecal space todeliver light to the sacral roots, and/or the cell bodies of the nerveslocated within the dorsal and ventral horns of the spine, and/or nerveganglia located near or about the sline, for optogenetic modulation ofneurons involved in bowel, bladder, and erectile function. Alternately,it may be inserted higher in the spinal column for pain controlapplications, such as those described elsewhere in this application.Either the linear or matrix array optarray(s) may be inserted into theanterior intrathecal anatomy to control motor neurons and/or into theposterior intrathecal anatomy to control sensory neurons. A singleoptical element may be illuminated for greater specificity, or multipleelements may be illuminated. FIG. 28 (1016) illustrates an alternativeview of an exemplary linear array.

The system may be tested at the time of implantation, or subsequent toit. The tests may provide for system configurations, such as which areasof the applicator are most effective, or efficacious, by triggeringdifferent light sources alone, or in combination, to ascertain theireffect on the patient. This may be utilized when a multi-element system,such as an array of LEDs, for example, or a multiple output couplingmethod is used. Such diagnostic measurements may be achieved by using animplanted electrode that resides on, in or near the applicator, or onethat was implanted elsewhere, as will be described in another section.Alternately, such measurements maybe made at the time of implantationusing a local nerve electrode for induced stimulation, and/or anelectrical probe to query the nerve impulses intraoperatively using adevice such as the Stimulator/Locator sold under the tradenameCHECKPOINT® from NDI and Checkpoint Surgical, Inc. to provide electricalstimulation of exposed motor nerves or muscle tissue and in turn locateand identify nerves as well to test their excitability. Once obtained,an applicator illumination configuration may be programmed into thesystem for optimal therapeutic outcome using an externalProgrammer/Controller (P/C) via a Telemetry Module (TM) into theController, or Processor/CPU of the system Housing (H), as are definedfurther below.

FIG. 29A illustrates the gross anatomical location of animplantation/installation configuration wherein a controller housing (H)is implanted adjacent the pelvis, and is operatively coupled (via thedelivery segment DS) to an applicator (A) positioned to stimulate one ormore of the sacral nerve roots.

FIG. 29B illustrates the gross anatomical location of animplantation/installation configuration wherein a controller housing (H)is implanted adjacent the pelvis, and is operatively coupled (via thedelivery segment DS) to an applicator (A) positioned to stimulate one ormore of the lumbar, thoracic, or cervical nerve roots, such as bythreading the delivery segment and applicator into the intrathecal spaceto reach the pertinent root anatomy.

The electrical connections for devices such as these where the lightsource is either embedded within, on, or located nearby to theapplicator, may be integrated into the applicators described herein.Materials like the product sold by NanoSonics, Inc. under the tradenameMetal Rubber™ and/or mc10's extensible inorganic flexible circuitplatform may be used to fabricate an electrical circuit on or within anapplicator. Alternately, the product sold by DuPont, Inc., under thetradename PYRALUX®, or other such flexible and electrically insulatingmaterial, like polyimide, may be used to form a flexible circuit;including one with a copper-clad laminate for connections. PYRALUX® insheet form allows for such a circuit to be rolled. More flexibility maybe afforded by cutting the circuit material into a shape that containsonly the electrodes and a small surrounding area of polyimide.

Such circuits then may be encapsulated for electrical isolation using aconformal coating. A variety of such conformal insulation coatings areavailable, including by way of non-limiting example, parlene(Poly-Para-Xylylene) and parlene-C (parylene with the addition of onechlorine group per repeat unit), both of which are chemically andbiologically inert. Silicones and polyurethanes may also be used, andmay be made to comprise the applicator body, or substrate, itself. Thecoating material can be applied by various methods, including brushing,spraying and dipping. Parylene-C is a bio-accepted coating for stents,defibrillators, pacemakers and other devices permanently implanted intothe body.

In a particular embodiment, biocompatible and bio-inert coatings may beused to reduce foreign body responses, such as that may result in cellgrowth over or around an applicator and change the optical properties ofthe system. These coatings may also be made to adhere to the electrodesand to the interface between the array and the hermetic packaging thatforms the applicator.

By way of non-limiting example, both parylene-C and poly(ethyleneglycol) (PEG, described herein) have been shown to be biocompatible andmay be used as encapsulating materials for an applicator. Bioinertmaterials non-specifically downregulate, or otherwise ameliorate,biological responses. An example of such a bioinert material for use inan embodiment of the present invention is phosphoryl choline, thehydrophilic head group of phospholipids (lecithin and sphingomyelin),which predominate in the outer envelope of mammalian cell membranes.Another such example is Polyethylene oxide polymers (PEO), which providesome of the properties of natural mucous membrane surfaces. PEO polymersare highly hydrophilic, mobile, long chain molecules, which may trap alarge hydration shell. They may enhance resistance to protein and cellspoliation, and may be applied onto a variety of material surfaces, suchas PDMS, or other such polymers. An alternate embodiment of abiocompatible and bioinert material combination for use in practicingthe present invention is phosphoryl choline (PC) copolymer, which may becoated on a PDMS substrate. Alternately, a metallic coating, such asgold or platinum, as were described earlier, may also be used. Suchmetallic coatings may be further configured to provide for a bioinertouter layer formed of self-assembled monolayers (SAMs) of, for example,D-mannitol-terminated alkanethiols. Such a SAM may be produced bysoaking the intended device to be coated in 2 mM alkanethiol solution(in ethanol) overnight at room temperature to allow the SAMs to formupon it. The device may then be taken out and washed with absoluteethanol and dried with nitrogen to clean it.

A variety of embodiments of light applicators are disclosed herein.There are further bifurcations that depend upon where the light isproduced (i.e., in or near the applicator vs. in the housing orelsewhere). FIGS. 30A and 30B illustrate these two configurations.

Referring to FIG. 30A, in a first configuration, light is generated inthe housing and transported to the applicator via the delivery segment.The delivery segment(s) may be optical waveguides, selected from thegroup consisting of round fibers, hollow waveguides, holey fibers,photonic bandgap devices, and/or slab configurations, as have describedpreviously. Multiple waveguides may also be employed for differentpurposes. As a non-limiting example, a traditional circularcross-section optical fiber may be used to transport light from thesource to the applicator because such fibers are ubiquitous and may bemade to be robust and flexible. Alternately, such a fiber may be used asinput to another waveguide, this with a polygonal cross-sectionproviding for regular tiling. Such waveguides have cross-sectionalshapes that pack together fully, i.e. they form an edge-to-edge tiling,or tessellation, by means of regular congruent polygons. That is, theyhave the property that their cross-sectional geometry allows them tocompletely fill (pack) a two-dimensional space. This geometry yields theoptical property that the illumination may be made to spatiallyhomogeneous across the face of such a waveguide. Complete homogeneity isnot possible with other geometries, although they may be made to havefairly homogeneous irradiation profiles nonetheless. For the presentapplication, a homogenous irradiation distribution may be utilizedbecause it may provide for uniform illumination of the target tissue.Thus, such regular-tiling cross-section waveguides may be useful. It isalso to be understood that this is a schematic representation and thatmultiple applicators and their respective delivery segments may beemployed. Alternately, a single delivery segment may service multipleapplicators. Similarly, a plurality of applicator types may also beemployed, based upon the clinical need.

Referring to the configuration of FIG. 30B, light is in the applicator.The power to generate the optical output is contained within the housingand is transported to the applicator via the delivery segment. It is tobe understood that this is a schematic representation and that multipleapplicators and their respective delivery segments may be employed.Similarly, a plurality of applicator types may also be employed.

The size(s) of these applicators may be dictated by the anatomy of thetarget tissue. By way of non-limiting example, a fluidic channelslab-type (or, equivalently, “slab-like”) applicator may be configuredto comprise a parallel array of 3 rectangular HRIP waveguides that are200 μm on a side, the applicator may be between 1-10 mm in width andbetween 5-100 mm in length, and provide for multiple output couplersalong the length of each channel waveguide to provide a distributedillumination of the target tissue.

The pertinent delivery segments may be optical waveguides, such asoptical fibers, in the case where the light is not generated in or nearthe applicator(s). Alternately, when the light is generated at or nearthe applicator(s), the delivery segments may be electrical wires. Theymay be further comprised of fluidic conduits to provide for fluidiccontrol and/or adjustment of the applicator(s). They may also be anycombination thereof, as dictated by the specific embodiment utilized, ashave been previously described.

Embodiments of the subject system may be partially, or entirely,implanted in the body of a patient. FIG. 31 illustrates this, whereinthe left hand side of the illustration schematically depicts thepartially implanted system, and the right hand side of the illustrationthe fully implanted device. The housing H may be implanted, carried, orworn on the body (B), along with the use of percutaneous feedthroughs orports for optical and/or electrical conduits that comprise the deliverysegments (various embodiments/denotations of DS, or “DSx”, as per theFigures) that connect to Applicator(s) A implanted to irradiate targettissue(s) N. In this exemplary embodiment, a Transcutaneous OpticalFeedthrough COFT may be coupled to the Delivery Segments affixed toHousing H, located in Extracorporeal Space ES, while Applicator A is inthe Intracorporeal Space IS along with Target Tissue N.

FIG. 70 shows an embodiment of a transcutaneous optical feedthrough, orport, comprising, by way of non-limiting example, an External DeliverySegment DSE, which in turn is routed through a seal, comprised of,External Sealing Element SSE that resides in the extracorporeal spaceES, and Internal Sealing Element SSI that resides in the intracorporealspace IS. These sealing elements may held together by means ofCompression Element COMPR to substantially maintain an infection-freeseal for Transcutaneous Optical Feedthrough COFT. Internal Seal SSI, maycomprise a medical fabric sealing surface along with a more rigid membercoupled thereto to more substantially impart the compressive force fromCompression Element COMPR when forming a percutaneous seal. The medicalfabric/textile may be selected from the list consisting of, by way ofnon-limiting examples; dacron, polyethylene, polypropylene, silicone,nylon, and PTFE. Woven and/or non-woven textiles may be used as acomponent of Internal Seal SSI. The fabric, or a component thereon, mayalso be made to elute compounds to modulate wound healing and improvethe character of the seal. Such compounds, by way of non-limitingexamples, may be selected from the list consisting of; VascularEndothelial Growth Factor (VEGF), glycosaminoglycans (Gags), and othercytokines. Applicable medical textiles may be available from vendors,such as Dupont and ATEX Technologies, for example. Delivery Segment DSmay be connected to the optical and/or electrical connections ofApplicator A, not shown for purposes of clarity, not shown. ExternalDelivery Segment DSE may be may be connected to the optical and/orelectrical output of Housing H, not shown for purposes of clarity. Thesurface of the patient, indicated in this example as Skin SKIN, mayoffer a natural element by way of the epidermis onto which the seal maybe formed. Details regarding the means of sealing External DeliverySegment DES, which passes through the Skin SKIN, to Compression ElementCOMPR are discussed elsewhere herein in regards to optical feedthroughswithin Housing H, such as are shown in FIGS. 73A-75.

FIGS. 71A-C show an alternate embodiment, wherein a plurality ofwaveguides, such as, but not limited to fibers, may be bundled togetherfor ease of capturing the light at input face INPUT FACE from a singlelight source, such as, by way of non-limiting examples, an LED or alaser and distributing it to a target tissue within the spine of apatient. The fibers, OFx may be connectorized at the input face INPUTFACE using circular ferrule connector CFC to form bundle BUNDLE. Thebundle may then be further dressed using mid-section retaining ferruleMSRF. It ma y then splay out into a one-dimensional array by utilizingFlat Retaining Ferrule FRF to dispose the array to shine light on orover a desired area(s), such as, by way of non-limiting example, theleft and right portions of the ventral spinal cord between L5 and L6 inorder to target the roots pudendal nerve, via lateral sections SB1 andSB2. The distal tips DFTx of the individual Optical Fibers OFx within aFiber Bundle FBx may be bent in the direction of desired illumination,or ground and coated to fire the light sideways, or have a opticalelement, such as, by way of non-limiting example, a mirror, lens, orprism, located distal to the output face to ultimately direct the lightto the target tissue, such as has been described elsewhere hereinregarding the optical output of applicators. The output ends DFTx maythen be encapsulated in a permanent transparent coating to hold them inthe desired position. Radio-opaque markings may be placed at, on, oralong the applicator to improve placement accuracy relative to thetarget tissue under fluoroscopic guidance.

A non-limiting example of how such a bundled fiber light deliverysegment may be constructed follows. All fibers at the input end of thebundle may be grouped together inside a temporary ferrule with aremovable adhesive. This ferrule may be round, rectangular, or othershape. One embodiment holds all fibers in a single layer between twoflat pieces of material, to be ground and polished at a desired angle,such as 45°. These angled ends of the fibers may be plated with areflective coating, as mentioned earlier. The fibers may then be routedthrough a removable length guide which holds each fiber or group offibers at individual lengths to create a range of lengths, such thatthey are spaced to fit the finished configuration of Applicator A.Optical Fibers OFx may be bundled, or re-bundled in another temporary orpermanent ferrule or other fixing member with a removable or permanentadhesive at or near their Output Ends DFTx. Output Ends DFTx may then beremoved from the length guide and Output Ends DFTx pulled tight and thenbundled in a third permanent circular, square, rectangular or othershaped ferrule with a permanent adhesive at the desired length. Thisoutput end may then be cut and polished. The fiber bundle may then beremoved from the distal ferrule and adhesive. At this point the distalends of the fibers should straighten and project linearly from the mostdistal ferrule at the range of lengths defined by the length guide. Theangled polished (and possibly coated) ends should all be made to pointin approximately the same direction, and/or at the same point in spacedepending upon the final configuration for Applicator A. The output endsmay then be encapsulated in a permanent transparent coating to hold themin the desired position using fixtures if necessary. Some non-limitingexamples of this coating are over-molded, cast or dipped silicone,polyimide tape with silicone or acrylic adhesive or two layers ofpolyethylene heat-welded together.

FIGS. 72A and 72B show an alternate embodiment of the present inventionin which the Applicator A is configured to encapsulate the lateralsections SB1 and SB2 containing Distal Tips DFTx of individual OpticalFibers OFx within Fiber Bundle FBx that comprises Delivery Segment DS.Optical Fibers OFx are separated within Fan Segment FAN-SEG and disposedat a Distal Segment DIST-SEG within Applicator A for treatment at thespine, or other such predominantly linear anatomical location, of apatient. Similar to FIG. 72A, FIG. 72B shows a more detailed view of theDistal Segment DIST-SEG, in which the addition of Encapsulant ENCAPS isshown with Distal Tips DFTx of individual Optical Fibers OFx.

FIGS. 73A AND 73B show an alternate embodiment of an implantable,hermetically sealed Housing H comprising an optical feed-through OFT,wherein Delivery Segment DSx may be coupled to Housing H. The systemfurther may comprise a configuration such that Delivery Segment DSx maybe coupled to Housing H via a plurality of electrical connections and atleast one optical connection via Connector C, which in this exemplaryembodiment is shown as a component of Delivery Segment DS, but alternateconfigurations are within the scope of the present invention. Also shownare hidden line views of the Housing H, Delivery Segment DSx, andConnector C that reveal details of an embodiment, such as Circuit BoardCBx, Light Source LSx, Optical Lens OLx, the proximal portion of theDelivery Segment DSx, and a Hermetic Barrier HBx. Light Source LSx maybe mounted to and electrical delivered thereto by Circuit Board CBx.Optical Lens OLx may be a sapphire rod lens that serves to transmitlight to Delivery Segment DSx.

FIG. 74 shows an enlarged view of the implantable Housing H and theoptical feed-through OFT, comprised of the Optical Lens OLx and theFlanged Seal FSx. In an exemplary embodiment, the outer cylindricalsurface of the sapphire lens may be coated with high purity gold, forexample, and brazed to a flanged seal, such as a titanium seal, in abrazing furnace. This may create a biocompatible hermetic connectionbetween Optical Lens OLx and the Flanged Seal FSx. The exemplarylens-seal combination may then be inserted into a hole in the outersurface of Housing H, which may also be comprised of titanium, andFlanged Seal FSx welded at least partially about the perimeter of acomplementary hole in Housing H. This may create a completelybiocompatible hermetically sealed assembly through which light fromLight Source LSx may be coupled from within Housing H and transmit lightoutside of Housing H for use by Delivery Segments DS, and/or anApplicator A for treatment at a target tissue, as has been describedelsewhere herein.

FIG. 75 shows an isometric view of an embodiment of the presentinvention, in which Light Source LSx may be at least partially opticallycoupled to fiber bundle FBx via Optical Lens OLx interposed between thetwo. Optically index-matched adhesive may be used to affix Optical LensOLx onto Light Source LSx directly. It should be understood that thelight source may be contained within a hermetically sealed implantablehousing, not shown for clarity, and that Optical Lens OLx crosses thewall of the hermetically sealed implantable Housing H wherein a portionof Optical Lens OLx resides within Housing H and another portion ofOptical Lens OLx resides outside of Housing H and is hermetically sealedaround at least a portion of its Outer Surface OS, and that a FiberBundle FB may reside outside the hermetically sealed implantable HousingH and may be coupled to Optical Lens OLx. For instance, if a singlesource Light Source LS is used, such as an LED, a bundle of 7 OpticalFibers OFx may be used to capture the output of Light Source LS, whichmay be, for example, a 1 mm×1 mm LED. Fiber Bundle FB may have an outerdiameter of 1 mm to assure that all Optical Fibers OFx are exposed tothe output of Light Source LS. Using fibers of 0.33 mm outer (cladding)diameter is the most efficient way of packing 7 fibers into a circularcross section using a hexagonal close-packed (HCP) configuration toapproximate a 1 mm diameter circle. The ultimate optical collectionefficiency will scale from the filling ratio, the square of the fibercore/cladding ratio, and in further proportion to the ratio of the fiberétendue to that of the LED output as the numerical apertures areconsidered. These sub-fibers, or sub-bundles as the case may be, may beseperated and further routed, trimmed, cut, polished, and/or lensed,depending upon the desired configuration. Brazing of Optical Lens OLxand the Flanged Seal FSx should be performed prior to the use ofadhesives.

Number Circular Square of Fibers Filling % Filling % 7 78 61 19 80 63 3781 63.5 55 81.5 64 85 82 64.5

The above table describes several different possibilities for couplinglight from a single source into a plurality of fibers (a bundle) in aspatially efficient manner. For circular fibers, the HCP configurationhas a maximum filling ratio of ˜90.7%. It should be understood that evenmore efficient bundles may be constructed using hexagonal or otherwiseshaped individual fibers and the Fiber Bundles FBx shown are merely forexemplary purposes. The plurality of fibers may be separated in tosmaller, more flexible sub-bundles. Fiber Bundles FBx may be adhesivelybonded together and/or housed within a sheath, not shown for clarity.Multiple smaller Optical Fibers OFx may be used to provide an ultimatelymore flexible Fiber Bundle FBx, and may be flexibly routed throughtortuous pathways to access target tissue. Additionally, Optical FibersOFx may be separated either individually or in sub groups to be routedto more than one target tissue site. For instance, if a seven fiberconstruct is used, these seven fibers may be routed to seven individualtargets. Similarly, if a 7×7 construction is used, the individualbundles of 7 fibers may be similarly routed to seven individual targetsand may be more flexible than the alternative 1×7 construct fiber bundleand hence routed to the target more easily.

FIG. 76A shows an embodiment of the present invention similar to thatshown in FIGS. 26-28, wherein an Applicator A is now configured forintrathecal spinal illumination, where a Pull Wire(s) PWx may be coupledto Biasing Segments BSx and Illumination Segments ISx, such thatApplicator A expands to fill the intrathecal space when force is appliedto pull the Pull Wire(s) PWx, Delivery Segments DSx separate to form FanSegment FAN-SEG. Pull Wire(s) PWx are then coupled to the distalsections of the Biasing Segments BSx at Distal Pivot Element BWDPx.Common Pivot CP is located at the distal-most end of the applicator.Proximal Pivot Element(s) BWPPx may serve to distribute the load fromPull Wire(s) PWx. Pull Wires PWx may be routed off axis, substantiallyat or near the Distal and/or Proximal locations of the Biasing orIllumination segments, BWDPx and BWPPx, respectively, such thatasymmetrical axial loads are placed on the segments, thereby imparting abending moment within the Biasing Segments BSx and Illumination SegmentsISx.

As configured, pulling on Pull Wire(s) PWx may cause the Light SourcesLSx located on or about Illumination Segment(s) ISx to move in onedirection and Biasing Segments BSx to move in the opposite direction, asshown in FIG. 76B. In this fashion Light Sources LSx are deployedagainst the spinal cord and the Biasing Segments BSx is placed againstthe wall of the spinal dura. This design may provide for the applicationof low and even pressure while accommodating the geometry of theintrathecal space, as is shown in the alternate view of this exemplaryconfiguration in FIG. 76B, where Biasing Segment(s) BSx are locateddorsally for illumination of the dorsal roots and ventrally forillumination of the ventral roots. The amount of movement, ortranslocation, of the Illumination Segments ISx and Biasing Segments BSxis proportional to the amount of Pull Wire(s) movement (and thereforeforce applied to the pull wire).

A structure may be comprised of a flexible material, such as silicone(as has been described elsewhere herein), which may be compressed viarolling or folding to fit through a small introducer, such as; anendoscope, a laproscope, a cannula, or a catheter. It may be furtherconfigured to expand and fit securely in the intradural space of thespinal column when removed from the introducer. The structure may have alocation for the mounting LEDs such that when the device is deployed inthe spinal column, the LED outputs being directed toward the targettissue. The structure may be fabricated of conductive wires or tracesand insulated so that the structure forms the circuitry used to powerthe LEDs. Additionally, control wires can be included so that thelocation of the LEDs may be adjusted relative to the location of thesecuring features. The entire structure may be shaped in 3 dimensions tominimize any pressure applied to the spinal cord or any other tissue.The structure may be shaped and or adjusted through control wires toplace a small amount of pressure holding the LEDs against the target tomaximize light transmission to the targeted cells within the targettissue.

FIG. 77 shows details regarding the therapeutic placement of theintrathecal Applicator A for illumination of the dorsal aspect of theSpinal Cord CORD, and illustrates the locations anatomical elementsFORAMEN, and Vertebral Body VB for anatomic reference.

FIG. 78 shows the exemplary embodiment of FIG. 76A & FIG. 76B in a statefor insertion into the intrathecal space of the patient, as describedearlier, where Pull Wires PWx are in their relaxed state, which maycause the individual segments of Applicator A, such as Bias Segments BSxand Illumination Segments ISx to be substantially adjacent, and/oroverlapped to present a minimal cross-sectional area. For the purposesof these embodiments, the term Lighting Segment may be used to describethe segment of the Applicator for illuminating the spine using eitherproximal or distal Light Sources LSx, (e.g. LEDs on the lightingsegment(s), and/or waveguides transmitting light from a remote lightsource).

FIG. 79 illustrates an embodiment of the present invention, wherein anApplicator A may be used to illuminate a target tissue N with using atleast one Light Source LSx. Light Source(s) LSx may be LEDs or laserdiodes. Light Source(s) LSx may be located at or adjacent to the targettissue, and reside at least partially within an Applicator A, and beelectrically connected by Delivery Segment(s) DS to their power supplyand controller that reside, for example, inside a Housing H.

FIG. 80 shows such an exemplary system configuration. In thisillustrative embodiment, a single strip of LEDs is encased in anoptically clear and flexible silicone, such as the low durometer,unrestricted grade implantable materials MED-4714 or MED4-4420 fromNuSil, by way of non-limiting examples. This configuration provides arelatively large surface area for the dissipation of heat. For example,a 0.2 mm×0.2 mm 473 nm wavelength LED, such as those used in the picoLEDdevices by Rohm, or the die from the Luxeon Rebel from Phillips, mayproduce about 1.2 mW of light. In the exemplary embodiment beingdescribed, there are 25 LEDs utilized, producing a total of about 30 mWof light, and in turn generate about 60 mW of heat. They are nominallybetween 30-50% efficient. The heat generated by the LEDs may bedissipated over the relatively large surface area afforded by thepresent invention of 15 mm², or a heat flux of 4 mW/mm² at the surfaceof Applicator A. Implantable (unrestricted) grade silicone has a thermalconductivity of about 0.82Wm⁻¹ K⁻¹, and a thermal diffusivity of about0.22 mm² s⁻¹ and distributing the heat over a larger area and/or volumeof this material decreases the peak temperature rise produced at thetissue surface.

FIG. 81 illustrates an alternate configuration of the embodiment of FIG.79, with the addition of a spiral, or helical design for Applicator A isutilized. Such a configuration may allow for greater exposure extent ofthe target tissue. This may also be useful to allow slight misplacementof the applicator with respect to the target tissue, if the longitudinalexposure length is greater than that intended for the target tissue andthe deployed location of Applicator A also subsumes the target tissue bya reasonable margin. A reasonable margin for most peripheralapplications is about ±2 mm. Applicator A must provide an inner diameter(ID) that is at least slightly larger than the outer diameter (OD) ofthe target tissue for the target tissue with Applicator A to moveaxially without undue stress. Slightly larger in the case of mostperipheral nerves may provide that the ID of Applicator A be 5-10%larger than the target tissue OD.

Fiber and or protective coverings on or containing a waveguide, such as,but not limited to optical fiber may be shaped to provide astrain-relieving geometry such that forces on the applicator are muchreduced before they are transmitted to the target tissue. By way ofnon-limiting example, shapes for a flexible fiber to reduce forces onthe target tissue include; serpentine, helical, spiral, dualnon-overlapping spiral (or “bowtie”), cloverleaf, or any combination ofthese.

FIG. 82A-82D illustrate a few of these different configurations in whichUndulations U are configured to create a strain relief section ofoptical waveguide Delivery Segment DS prior to its connection toApplicator A via Connector C. FIG. 82A illustrates a Serpentine sectionof Undulations U for creating a strain relief section within DeliverySegment DS and/or Applicator A. FIG. 82B illustrates a Helical sectionof Undulations U for creating a strain relief section within DeliverySegment DS and/or Applicator A. FIG. 82C illustrates a Spiral section ofUndulations U for creating a strain relief section within DeliverySegment DS and/or Applicator A. FIG. 82D illustrates a Bowtie section ofUndulations U for creating a strain relief section within DeliverySegment DS and/or Applicator A. Target Tissue resides within Applicatorin these exemplary embodiments, but other configurations, as have beendescribed elsewhere herein, are also within the scope of the presentinvention.

FIG. 83 shows an alternate embodiment, wherein Applicator A may beconfigured such that it is oriented at an angle relative the DeliverySegment DS, and not normal to it as was illustrated in the earlierexemplary embodiments. Such an angle might be required, for example, inorder to accommodate anatomical limitations, such as the target tissueresiding in a crevice or pocket, as may the case for certain peripheralnerves. Another bend, or Undulation U, in either the Delivery Segment DSor in an element of Applicator A, such as an output coupler, as has beendescribed elsewhere herein, may be utilized to create the angle.

In an alternate embodiment, an optical feature may be incorporated intothe system at the distal end of the Delivery Segment DS, or the proximalend of the optical input of Applicator A to reflect the light an anglerelative to the direction of the fiber to achieve the angle.

Plastic optical fiber such as 100 μm core diameter ESKA SK-10 fromMitsubishi may be routed and/or shaped in a jig and then heat-set toform Undulations U directly. Alternately, a covering may be used overthe waveguide, and that covering may be fabricated to create UndulationsU in the waveguide indirectly. An alternate exemplary plastic fiberwaveguide may be constructed from a PMMA (n=1.49) core material with acladding of THV (n−1.35) to provide an NA of 0.63. A polyethylene tube,such as, PE10 from Instech Solomon, may be used as a cover, shaped in ajig and heat-set to create Undulations U while using a silica opticalfiber within the tube. Heat-setting for these two exemplary embodimentsmay be accomplished by routing the element to be shaped in a jig or toolto maintain the desired shape, or one approximating it, and then heatingthe assembly in an oven at 70° C. for 30 minutes. Alternately, the bendsmay be created in more gradual steps, such that only small bends aremade at each step and the final heating (or annealing) provides thedesired shape. This approach may better assure that no stress-inducedoptical changes are ingendered, such as refractive index variations,which might result in transmission loss. Although optical fiber has beendiscussed in the previous examples, other delivery segment andapplicator configurations are within the scope of the present invention.

Light transmission through tissue such as skin is diffusive, andscattering the dominant process. Scattering diminishes thedirectionality and brightness of light illuminating tissue. Thus, theuse of highly directional and/or bright sources is rendered moot. Thismay limit the depth in tissue that a target may be affected. An in-vivolight collector may used within the tissue of a patient in cases wherestraightforward transcutaneous illumination cannot be used to adequatelyirradiate a target due to irradiance reduction, and a fully implantedsystem may be deemed too invasive.

In one embodiment, an at least partially implanted system for collectinglight from an external source may be placed in-vivo and/or in-situwithin the skin of a patient to capture and transmit light between theexternal light source and an implanted applicator. Such applicators havebeen described elsewhere herein.

Alternately, an at least partially implanted system for collecting lightfrom an external source may be placed in-vivo and/or in-situ within theskin of a patient to capture and transmit light between the externallight source and direct it to the target tissue directly, without theuse of a separate applicator.

The light collection element of the system may be constructed, forexample, from a polymer material that has an outer layer of a nominallydifferent index of refraction than that of the body or core material,such as is done in fiber optics. While the index of refraction of skinand other tissues is about equal to that of water, corresponding to arange of 1.33-1.40 in the visible spectrum, and would provide afunctional cladding that may yield an NA as high as 0.56 when PMMA isused is the unclad core material. However, native chromophores withintissues such as skin that may be avid absorbers of the light from theexternal light source, especially visible light. Examples of such nativechromophores are globins (e.g. oxy-, deoxy-, and met-hemoglobin),melanins (e.g. neuro-, eu-, and pheo-melanin), and xanthophylls (e.g.carotenol fatty acid esters). The evanescent wave present in aninsufficiently clad or unclad collection device may be coupled intoabsorption by these native pigments that potentially causes unintendedand/or collateral heating that not only diminishes the amount of lightconducted to the target, but also may create a coating on the collectorthat continually degrades its performance. For example, there may bemelanin resident at the dermal-epidermal junction, and blood resident inthe capillary bed of the skin.

In one embodiment, the depth of the surface of the implantable lightconductor is placed between 100 and 1000 μm beneath the tissue surface.In the case of cutaneous implantation, this puts that surface below theepidermis.

The implantable light collector/conductor may be made of polymeric,glass, or crystalline material. Some non-limiting examples are; PMMA,Silicones, such as MED-4714 or MED4-4420 from NuSil, PDMS, andHigh-Refractive-Index Polymers (HRIPs), as are described elsewhereherein.

A cladding layer may also be used on the implantable light collector toimprove reliability, robustness and overall performance. By way ofnon-limiting example, THV (a low index fluoropolymer blend), Fluorinatedethylene propylene (FEP), and/or polymethylpentene may be used toconstruct cladding layers about a core material. These materials arebiocompatible and possess relatively low indices of refraction(n=1.35-1.4). Thus, they provide for light collection over a widenumerical aperture (NA).

In addition to the use of a cladding layer on the implantable lightconductor/collector, a coating may be disposed to the outer surface ofthe conductor/collector to directly confine the light within theconductor, and/or to keep the maintain the optical quality of the outersurface to avoid absorption by native chromophores in the tissue at ornear the outer surface of the collector because the evanescent wavepresent in a waveguide may still interact with the immediateenvironment. Such coating might be, for example, metallic coatings, suchas, Gold, Silver, Rhodium, Platinum, Aluminum. A dielectric coating mayalso be used. Examples being; SiO₂, Al₂O₃ for protecting a metalliccoating, or a layered dielectric stack coating to improve reflectivity,or a simple single layer coating to do likewise, such as quarter-wavethickness of MgF₂.

Alternately, the outer surface of the implantable light collector may beconfigured to utilize a pilot member for the introduction of the deviceinto the tissue. This pilot member may be configured to be a cuttingtool and/or dilator, from which the implantable light conductor may beremovably coupled for implantation.

Implantation may be performed, by way of non-limiting example, usingpre-operative and/or intra-operative imaging, such as radiography,fluoroscopy, ultrasound, magnetic resonance imaging (MRI), computedtomography (CT), optical imaging, microscopy, confocal microscopy,endoscopy, and optical coherence tomography (OCT).

Alternately, the pilot member may also form a base into which theimplantable light collector is retained while implanted. As such, thepilot member may be a metal housing that circumscribes the outer surfaceof the implantable light collector and provides at least a nominallysheltered environment. In such cases replacement of the light collectormay be made easier by leaving in place the retaining member (as theimplanted pilot member may be known) and exchanging the light collectoronly. This may be done, for example, in cases where chronic implantationis problematic and the optical quality and/or efficiency of the lightcollector diminishes.

Alternately, the outer surface of the implantable collector may be mademore bioinert by utilizing coatings of: Gold or Platinum, parylene-C,poly(ethylene glycol) (PEG), phosphoryl choline, Polyethylene oxidepolymer, self-assembled monolayers (SAMs) of, for example,D-mannitol-terminated alkanethiols, as has been described elsewhereherein.

The collection element may be comprised of, by way of non-limitingexample, an optical fiber or waveguide, a lightpipe, or plurality ofsuch elements. For example, considering only scattering effects, asingle 500 μm diameter optical fiber with an intrinsic numericalaperture (NA) of 0.5 that is located 300 μm below the skin surface maybe able to capture at most about 2% of the light from a 01 mm beam ofcollimated light incident upon the skin surface. Thus, a 1 W sourcepower may be required in order to capture 20 mW, and require a surfaceirradiance of 1.3 W/mm². This effect improves additively for each suchfiber included in the system. For example, 4 such fibers may lower thesurface incident optical power required by the same factor of 4 andstill capture 20 mW. Of course, this does not increase the deliveredbrightness at the target, but may provide for more power to be deliveredand distributed at the target, such as might be done in circumferentialillumination. It should be known that it is a fundamental law of physicsthat brightness cannot be increased without adding energy to a system.Multiple fibers, such as those described, may be used to supply light toan applicator via multiple delivery segments, as are described elsewhereherein.

Larger numbers of light collecting elements, such as the optical fiberwaveguides described in the embodiments above are also within the scopeof the present invention.

Similar to the embodiment of FIG. 38, an alternate embodiment is shownin FIG. 84. Light Rays LR from External Light Source ELS are shown inthe illustrative exemplary embodiment to exit External Light Source ELS,encounter External Boundary EB (such as the skin's stratum corneumand/or epidermis and subsequently traverse the Dermal-Epidermal JunctionDEJ) to reach the proximal surface of Implantable Light Collector PLS,where the proximal collection surface is divided into individualsections that each provide input for waveguides and/or delivery segmentsDSx that are operatively coupled to an Applicator A in order toilluminate target tissue N.

FIG. 85 illustrates an alternate embodiment similar to that of FIG. 84,where Implantable Light Collector PLS is not subdivided into separatesections, but instead supplies light to Applicator A via a single inputchannel. Delivery Segments DSx are not shown, but may be utilized in afurther embodiment.

Surface cooling techniques and apparatus may be used in furtherembodiments of the present invention to mitigate the risk of collateralthermal damage that may be caused by optical absorption by the melaninlocated at the dermal-epidermal junction. Basic skin-cooling approacheshave been described elsewhere. Such as, by way on non-limiting example,those described by U.S. Pat. Nos. 5,486,172; 5,595,568; and 5,814,040;which are incorporated herein in their entirety.

FIG. 86 illustrates an alternate embodiment of the present inventionsimilar to that of FIG. 85, but with the addition of Skin CoolingElement SCE. Skin Cooling Element SCE is shown in direct contact withthe skin surface, but need not be, as has been described in theaforementioned references immediately above. Similar to External LightSource ELS, Skin Cooling Element SCE may also be connected to a systemcontroller and power supply. The user may program the parameters of SkinCooling Element SCE to improve comfort and efficacy by adjusting theamount and/or temperature of the cooling, as well as its duration andtiming relative to the illumination light from External Light SourceELS. External is understood to be equivalent to extracorporeal.

In an alternate embodiment, a tissue clearing agent, such as thosedescribed elsewhere herein, may be used to improve the transmission oflight through tissue for collection by an implanted light collectiondevice. The following tissue clearing agents may be used, by way ofnon-limiting examples; glycerol, polypropylene glycol-based polymers,polyethylene glycol-based polymers (such as PEG200 and PEG400),polydimethylsiloxane, 1,4-butanediol, 1,2-propanediol, certainradiopaque x-ray contrast media (such as Reno-DIP, Diatrizoatemeglumine). For example, topical application of PEG-400 and Thiazone ina ratio of 9:1 for between 15-60 minutes may be used to decrease thescattering of human skin to improve the overall transmission of lightvia an implantable light collector.

Referring to FIG. 32, a block diagram is depicted illustrating variouscomponents of an example implantable housing H. In this example,implantable stimulator includes processor CPU, memory MEM, power supplyPS, telemetry module TM, antenna ANT, and the driving circuitry DC foran optical stimulation generator (which may or may not include a lightsource, as is described elsewhere herein). The Housing H is coupled toone Delivery Segments DSx, although it need not be. It may be amulti-channel device in the sense that it may be configured to includemultiple optical paths (e.g., multiple light sources and/or opticalwaveguides or conduits) that may deliver different optical outputs, someof which may have different wavelengths. More or less delivery segmentsmay be used in different implementations, such as, but not limited to,one, two, five or more optical fibers and associated light sources maybe provided. The delivery segments may be detachable from the housing,or be fixed.

Memory (MEM) may store instructions for execution by Processor CPU,optical and/or sensor data processed by sensing circuitry SC, andobtained from sensors both within the housing, such as battery level,discharge rate, etc., and those deployed outside of the Housing (H),possibly in Applicator A, such as optical and temperature sensors,and/or other information regarding therapy for the patient. Processor(CPU) may control Driving Circuitry DC to deliver power to the lightsource (not shown) according to a selected one or more of a plurality ofprograms or program groups stored in Memory (MEM). The Light Source maybe internal to the housing H, or remotely located in or near theapplicator (A), as previously described. Memory (MEM) may include anyelectronic data storage media, such as random access memory (RAM),read-only memory (ROM), electronically-erasable programmable ROM(EEPROM), flash memory, etc. Memory (MEM) may store program instructionsthat, when executed by Processor (CPU), cause Processor (CPU) to performvarious functions ascribed to Processor (CPU) and its subsystems, suchas dictate pulsing parameters for the light source.

Electrical connections may be through Housing H via an ElectricalFeedthrough EFT, such as, by way of non-limiting example, The SYGNUS®Implantable Contact System from Bal-SEAL.

In accordance with the techniques described in this disclosure,information stored in Memory (MEM) may include information regardingtherapy that the patient had previously received. Storing suchinformation may be useful for subsequent treatments such that, forexample, a clinician may retrieve the stored information to determinethe therapy applied to the patient during his/her last visit, inaccordance with this disclosure. Processor CPU may include one or moremicroprocessors, digital signal processors (DSPs), application-specificintegrated circuits (ASICs), field-programmable gate arrays (FPGAs), orother digital logic circuitry. Processor CPU controls operation ofimplantable stimulator, e.g., controls stimulation generator to deliverstimulation therapy according to a selected program or group of programsretrieved from memory (MEM). For example, processor (CPU) may controlDriving Circuitry DC to deliver optical signals, e.g., as stimulationpulses, with intensities, wavelengths, pulse widths (if applicable), andrates specified by one or more stimulation programs. Processor (CPU) mayalso control Driving Circuitry (DC) to selectively deliver thestimulation via subsets of Delivery Segments (DSx), and with stimulationspecified by one or more programs. Different delivery segments (DSx) maybe directed to different target tissue sites, as was previouslydescribed.

Telemetry module (TM) may include, by way of non-limiting example, aradio frequency (RF) transceiver to permit bi-directional communicationbetween implantable stimulator and each of a clinician programmer moduleand/or a patient programmer module (generically a clinician or patientprogrammer, or “C/P”). A more generic form is described above inreference to FIG. 3 as the input/output (I/O) aspect of a controllerconfiguration (P/C). Telemetry module (TM) may include an Antenna (ANT),of any of a variety of forms. For example, Antenna (ANT) may be formedby a conductive coil or wire embedded in a housing associated withmedical device. Alternatively, antenna (ANT) may be mounted on a circuitboard carrying other components of implantable stimulator or take theform of a circuit trace on the circuit board. In this way, telemetrymodule (TM) may permit communication with a programmer (C/P). Given theenergy demands and modest data-rate requirements, the Telemetry systemmay be configured to use inductive coupling to provide both telemetrycommunications and power for recharging, although a separate rechargingcircuit (RC) is shown in FIG. 32 for explanatory purposes. An alternateconfiguration is shown in FIG. 33.

Referring to FIG. 33, a telemetry carrier frequency of 175 kHz alignswith a common ISM band and may use on-off keying at 4.4 kbps to staywell within regulatory limits. Alternate telemetry modalities arediscussed elsewhere herein. The uplink may be an H-bridge driver acrossa resonant tuned coil. The telemetry capacitor, C1, may be placed inparallel with a larger recharge capacitor, C2, to provide a tuning rangeof 50-130 kHz for optimizing the RF-power recharge frequency. Due to thelarge dynamic range of the tank voltage, the implementation of theswitch, S1, employs a nMOS and pMOS transistor connected in series toavoid any parasitic leakage. When the switch is OFF, the gate of pMOStransistor is connected to battery voltage, VBattery, and the gate ofnMOS is at ground. When the switch is ON, the pMOS gate is at negativebattery voltage, −VBattery, and the nMOS gate is controlled by chargepump output voltage. The ON resistance of the switch is designed to beless than 5Ω to maintain a proper tank quality factor. A voltagelimiter, implemented with a large nMOS transistor, may be incorporatedin the circuit to set the full wave rectifier output slightly higherthan battery voltage. The output of the rectifier may then charge arechargeable battery through a regulator.

FIG. 34 relates to an embodiment of the Driving Circuitry DC, and may bemade to a separate integrated circuit (or “IC”), or application specificintegrated circuit (or “ASIC”), or a combination of them.

The control of the output pulse train, or burst, may be managed locallyby a state-machine, as shown in this non-limiting example, withparameters passed from the microprocessor. Most of the designconstraints are imposed by the output drive DAC. First, a stable currentis required to reference for the system. A constant current of 100 nA,generated and trimmed on chip, is used to drive the reference currentgenerator, which consists of an R-2Rbased DAC to generate an 8-bitreference current with a maximum value of 5 A. The reference current isthen amplified in the current output stage with the ratio of R_(o) andR_(ref), designed as a maximum value of 40. An on-chipsense-resistor-based architecture was chosen for the current outputstage to eliminate the need to keep output transistors in saturation,reducing voltage headroom requirements to improve power efficiency. Thearchitecture uses thin-film resistors (TFRs) in the output drivermirroring to enhance matching. To achieve accurate mirroring, the nodesX and Y may be forced to be the same by the negative feedback of theamplifier, which results in the same voltage drop on R_(o) and R_(ref).Therefore, the ratio of output current, I_(o), and the referencecurrent, Ire_(f), equals to the ratio of and R_(ref) and R_(o).

The capacitor, C, retains the voltage acquired in the precharge phase.When the voltage at Node Y is exactly equal to the earlier voltage atNode X, the stored voltage on C biases the gate of P2 properly so thatit balances I_(bias). If, for example, the voltage across R_(o) is lowerthan the original R_(ref)voltage, the gate of P2 is pulled up, allowingI_(bias) to pull down on the gate on P1, resulting in more current toR_(o). In the design of this embodiment, charge injection is minimizedby using a large holding capacitor of 10 pF. The performance may beeventually limited by resistor matching, leakage, and finite amplifiergain. With 512 current output stages, the optical stimulation IC maydrive two outputs for activation and inhibition (as shown in FIG. 34)with separate sources, each delivering a maximum current of 51.2 mA.

Alternatively, if the maximum back-bias on the optical element canwithstand the drop of the other element, then the devices can be drivenin opposite phases (one as sinks, one as sources) and the maximumcurrent exceeds 100 mA. The stimulation rate can be tuned from 0.153 Hzto 1 kHz and the pulse or burst duration(s) can be tuned from 100s to 12ms. However, the actual limitation in the stimulation output pulse-traincharacteristic is ultimately set by the energy transfer of the chargepump, and this generally should be considered when configuring thetherapeutic protocol.

The Housing H (or applicator, or the system via remote placement) mayfurther contain an accelerometer to provide sensor input to thecontroller resident in the housing. This may be useful for modulationand fine control of a hypertension device, for example, or forregulation of a pacemaker. Remote placement of an accelerometer may bemade at or near the anatomical element under optogenetic control, andmay reside within the applicator, or nearby it. In times of notabledetected motion, the system may alter it programming to accommodate thepatient's intentions and provide more or less stimulation and/orinhibition, as is required for the specific case at hand.

The Housing H may still further contain a fluidic pump (not shown) foruse with the applicator, as was previously described herein.

External programming devices for patient and/or physician can be used toalter the settings and performance of the implanted housing. Similarly,the implanted apparatus may communicate with the external device totransfer information regarding system status and feedback information.This may be configured to be a PC-based system, or a stand-alone system.In either case, the system generally should communicate with the housingvia the telemetry circuits of Telemetry Module (TM) and Antenna (ANT).Both patient and physician may utilize controller/programmers (C/P) totailor stimulation parameters such as duration of treatment, opticalintensity or amplitude, pulse width, pulse frequency, burst length, andburst rate, as is appropriate.

Once the communications link (CL) is established, data transfer betweenthe MMN programmer/controller and the housing may begin. Examples ofsuch data are:

1. From housing to controller/programmer:

-   -   a. Patient usage    -   b. Battery lifetime    -   c. Feedback data        -   i. Device diagnostics (such as direct optical transmission            measurements by an emitter-opposing photosensor)

2. From controller/programmer to housing:

-   -   a. Updated illumination level settings based upon device        diagnostics    -   b. Alterations to pulsing scheme    -   c. Reconfiguration of embedded circuitry        -   i. such as field programmable gate array (FPGA), application            specific integrated circuit (ASIC), or other integrated or            embedded circuitry

By way of non-limiting examples, near field communications, either lowpower and/or low frequency; such as ZigBee, may be employed fortelemetry. The tissue(s) of the body have a well-defined electromagneticresponse(s). For example, the relative permittivity of muscledemonstrates a monotonic log-log frequency response, or dispersion.Therefore, it is advantageous to operate an embedded telemetry device inthe frequency range of ≤1 GHz. In 2009 (and then updated in 2011), theUS FCC dedicated a portion of the EM Frequency spectrum for the wirelessbiotelemetry in implantable systems, known as The Medical DeviceRadiocommunications Service (known as “MedRadio”). Devices employingsuch telemetry may be known as “medical micropower networks” or “MMN”services. The currently reserved spectra are in the 401-406, 413-419,426-432, 438-444, and 451-457 MHz ranges, and provide for theseauthorized bandwidths:

-   -   401-401.85 MHz: 100 kHz    -   401.85-402 MHz: 150 kHz    -   402-405 MHz: 300 kHz    -   405-406 MHz: 100 kHz    -   413-419 MHz: 6 MHz    -   426-432 MHz: 6 MHz    -   438-444 MHz: 6 MHz    -   451-457 MHz: 6 MHz

The rules do not specify a channeling scheme for MedRadio devices.However, it should be understood that the FCC stipulates that:

-   -   MMNs should not cause harmful interference to other authorized        stations operating in the 413-419 MHz, 426-432 MHz, 438-444 MHz,        and 451-457 MHz bands.    -   MMNs must accept interference from other authorized stations        operating in the 413-419 MHz, 426-432 MHz, 438-444 MHz, and        451-457 MHz bands.    -   MMN devices may not be used to relay information to other        devices that are not part of the MMN using the 413-419 MHz,        426-432 MHz, 438-444 MHz, and 451-457 MHz frequency bands.    -   An MMN programmer/controller may communicate with a        programmer/controller of another MMN to coordinate sharing of        the wireless link.    -   Implanted MMN devices may only communicate with the        programmer/controller for their MMN.    -   An MMN implanted device may not communicate directly with        another MMN implanted device.    -   An MMN programmer/controller can only control implanted devices        within one patient.

Interestingly, these frequency bands are used for other purposes on aprimary basis such as Federal government and private land mobile radios,Federal government radars, and remote broadcast of radio stations. Ithas recently been shown that higher frequency ranges are also applicableand efficient for telemetry and wireless power transfer in implantablemedical devices.

An MMN may be made not to interfere or be interfered with by externalfields by means of a magnetic switch in the implant itself. Such aswitch may be only activated when the MMN programmer/controller is inclose proximity to the implant. This also provides for improvedelectrical efficiency due to the restriction of emission only whentriggered by the magnetic switch. Giant Magnetorestrictive (GMR) devicesare available with activation field strengths of between 5 and 150Gauss. This is typically referred to as the magnetic operate point.There is intrinsic hysteresis in GMR devices, and they also exhibit amagnetic release point range that is typically about one-half of theoperate point field strength. Thus, a design utilizing a magnetic fieldthat is close to the operate point will suffer from sensitivities to thedistance between the housing and the MMN programmer/controller, unlessthe field is shaped to accommodate this. Alternately, one may increasethe field strength of the MMN programmer/controller to provide forreduced sensitivity to position/distance between it and the implant. Ina further embodiment, the MMN may be made to require a frequency of themagnetic field to improve the safety profile and electrical efficiencyof the device, making it less susceptible to errant magnetic exposure.This can be accomplished by providing a tuned electrical circuit (suchas an L-C or R-C circuit) at the output of the switch.

Alternately, another type of magnetic device may be employed as aswitch. By way of non-limiting example, a MEMS device may be used. Acantilevered MEMS switch may be constructed such that one member of theMEMS may be made to physically contact another aspect of the MEMS byvirtue of its magnetic susceptibility, similar to a miniaturizedmagnetic reed switch. The suspended cantilever may be made to bemagnetically susceptible by depositing a ferromagnetic material (suchas, but not limited to Ni, Fe, Co, NiFe, and NdFeB) atop the end of thesupported cantilever member. Such a device may also be tuned by virtueof the cantilever length such that it only makes contact when theoscillations of the cantilever are driven by an oscillating magneticfield at frequencies beyond the natural resonance of the cantilever.

Alternately, an infrared-sensitive switch might be used. In thisembodiment of this aspect of the present invention, a photodiode orphotoconductor may be exposed to the outer surface of the housing and aninfrared light source used to initiate the communications link for theMMN. Infrared light penetrates body tissues more readily than visiblelight due to its reduced scattering. However, water and other intrinsicchromophores have avid absorption, with peaks at 960, 1180, 1440, and1950 nm, as are shown in the spectra of FIG. 35 (1018), where the waterspectrum runs form 700-2000 nm and that of adipose tissue runs from600-1100 nm.

However, the penetration depth in tissue is more influenced by itsscattering properties, as shown in the spectrum of FIG. 36 (1020), whichdisplays the optical scattering spectrum for human skin, including theindividual components from both Mie (elements of similar size to thewavelength of light) and Rayleigh (elements of smaller size than thewavelength of light) scattering effects.

This relatively monotonic reduction in optical scattering far outweighsabsorption, when the abovementioned peaks are avoided. Thus, an infrared(or near-infrared) transmitter operating within the range of 800-1300 nmmay be preferred. This spectral range is known as the skin's “opticalwindow.”

Such a system may further utilize an electronic circuit, such as thatshown in FIG. 37 (1022), for telemetry, and not just a sensing switch.Based upon optical signaling, such a system may perform at high datathroughput rates.

Generically, the signal-to-noise ratio (SNR) of a link is defined as,

${SNR}_{i} = {\frac{I_{s}}{I_{N}} = \frac{P_{s}R}{I_{N_{elec}} + {P_{N_{amb}}R}}}$

where I_(s) and I_(N) are the photocurrents resulting from incidentsignal optical power and photodiode noise current respectively, P_(s) isthe received signal optical power, R is the photodiode responsivity(A/W), I_(Nelec) is the input referred noise for the receiver andP_(Namb) is the incident optical power due to interfering light sources(such as ambient light).P_(s) can be further defined as

P _(s)=∫_(A) _(T) P _(Tx) J _(Rx) _(λ) η_(λ) dA

where P_(Tx) (W) is the optical power of the transmitted pulse, J_(Rx)_(λ) (cm⁻²) is the tissue's optical spatial impulse response flux atwavelength λ, η_(λ) is an efficiency factor (η_(λ)≤1) accounting for anyinefficiencies in optics/optical filters at λ and A_(T)represents thetissue area over which the receiver optics integrate the signal.

The abovementioned factors that affect the total signal photocurrent andtheir relationship to system level design parameters include emitterwavelength, emitter optical power, tissue effects, lens size,transmitter-receiver misalignment, receiver noise, ambient lightsources, photodiode responsivity, optical domain filtering, receiversignal domain filtering, line coding and photodiode and emitterselection. Each of these parameters can be independently manipulated toensure that the proper signal strength for a given design will beachieved.

Most potentially-interfering light sources have signal power thatconsists of relatively low frequencies (e.g. Daylight: DC; Fluorescentlights: frequencies up to tens or hundreds of kilohertz), and cantherefore be rejected by using a high-pass filter in the signal domainand using higher frequencies for data transmission.

The emitter may be chosen from the group consisting of, by way ofnon-limiting example, a VCSEL, an LED, a HCSEL. VCSELs are generallyboth higher brightness and more energy efficient than the other sourcesand they are capable of high-frequency modulation. An example of such alight source is the device sold under the model identifier “HFE4093-342” from Finisar, Inc., which operates at 860 nm and provides ≤5mW of average power. Other sources are also useful, as are a variety ofreceivers (detectors). Some non-limiting examples are listed in thefollowing table.

820-850 mm Agilent HFBR-1412 Agilent HFBR-2412 Agilent HFBR-1416 AgilentHFBR-2416 Hamamatsu L1915 Hamamatsu GT4176 Hamamatsu L5128 HamamatsuL5871 Hamamatsu L6486    950 nm Infineon SFH 4203 Infineon SFH 203Infineon SFH 4301 Infineon SFH 5400 Infineon SFH 4502 Infineon SFH 5440Infineon SFH 4503 Infineon SFH5441   1300 nm Agilent HFBR-1312 AgilentHFBR-2316 Hamamatsu L7866 Hamamatsu L7850

Alignment of the telemetry emitter to receiver may be improved by usinga non-contact registration system, such as an array of coordinatedmagnets with the housing that interact with sensors in thecontroller/programmer to provide positional information to the user thatthe units are aligned. In this way, the overall energy consumption ofthe entire system may be reduced.

Although glycerol and polyethylene glycol (PEG) reduce opticalscattering in human skin, their clinical utility has been very limited.Penetration of glycerol and PEG through intact skin is very minimal andextremely slow, because these agents are hydrophilic and penetrate thelipophilic stratum corneum poorly. In order to enhance skin penetration,these agents need to be either injected into the dermis or the stratumcorneum has to be removed, mechanically (e.g., tape stripping, lightabrasion) or thermally (e.g., erbium: yttrium-aluminum-garnet (YAG)laser ablation), etc. Such methods include tape stripping, ultrasound,iontophoresis, electroporation, microdermabrasion, laser ablation,needle-free injection guns, and photomechanically driven chemical waves(such as the process known as “optoporation”). Alternately, microneedlescontained in an array or on a roller (such as the Dermaroller®micro-needling device) may be used to decrease the penetration barrier.The Dermaroller® micro-needling device is configured such that each ofits 192 needles has a 70 μm diameter and 500 μm height. Thesemicroneedles are distributed uniformly atop a 2 cm wide by 2 cm diametercylindrical roller. Standard use of the microneedle roller typicallyresults in a perforation density of 240 perforations/cm² after 10 to 15applications over the same skin area. While such microneedle approachesare certainly functional and worthwhile, clinical utility would beimproved if the clearing agent could simply be applied topically ontointact skin and thereafter migrate across the stratum corneum andepidermis into the dermis. Food and Drug Administration (FDA) approvedlipophilic polypropylene glycol-based polymers (PPG) and hydrophilicPEG-based polymers, both with indices of refraction that closely matchthat of dermal collagen (n=1.47) are available alone and in a combinedpre-polymer mixture, such as polydimethylsiloxane (PDMS). PDMS isoptically clear, and, in general, is considered to be inert, non-toxicand non-flammable. It is occasionally called dimethicone and is one ofseveral types of silicone oil (polymerized siloxane), as was describedin detail in an earlier section. The chemical formula for PDMS isCH₃[Si(CH₃)₂O]_(n)Si(CH₃)₃, where n is the number of repeating monomer[SiO(CH₃)₂] units. The penetration of these optical clearing agents intoappropriately treated skin takes about 60 minutes to achieve a highdegree of scattering reduction and commensurate optical transportefficiency. With that in mind, a system utilizing this approach may beconfigured to activate its illumination after a time sufficient toestablish optical clearing, and in sufficient volume to maintain itnominally throughout or during the treatment exposure. Alternately, thepatient/user may be instructed to treat their skin a sufficient timeprior to system usage.

Alternately, the microneedle roller may be configured with the additionof central fluid chamber that may contain the tissue clearing agent,which is in communication with the needles. This configuration mayprovide for enhanced tissue clearing by allowing the tissue clearingagent to be injected directly via the microneedles.

A compression bandage-like system could push exposed emitters and/orapplicators into the tissue containing a subsurface optogenetic targetto provide enhanced optical penetration via pressure-induced tissueclearing in cases where the applicator is worn on the outside of thebody; as might be the case with a few of the clinical indicationsdescribed herein, like micromastia, erectile dysfunction, andneuropathic pain. This configuration may also be combined with tissueclearing agents for increased effect. The degree of pressure tolerableis certainly a function of the clinical application and the site of itsdisposition. Alternately, the combination of light source compressioninto the target area may also be combined with an implanted deliverysegment, or delivery segments, that would also serve to collect thelight from the external source for delivery to the applicator(s). Suchan example is shown in FIG. 38, where External Light Source PLS (whichmay the distal end of a delivery segment, or the light source itself) isplaced into contact with the External Boundary EB of the patient. ThePLS emits light into the body, which it may be collected by CollectionApparatus CA, which may be a lens, a concentrator, or any other means ofcollecting light, for propagation along Trunk Waveguide TWG, which may abundle of fibers, or other such configuration, which then bifurcatesinto separate interim delivery segments BNWGx, that in turn deliver thelight to Applicators Ax that are in proximity to Target N.

FIG. 87 illustrates an embodiment, where an external charging device ismounted onto clothing for simplified use by a patient, comprising aMounting Device MOUNTING DEVICE, which may be selected from the groupconsisting of, but not limited to: a vest, a sling, a strap, a shirt,and a pant. Mounting Device MOUNTING DEVICE further comprising aWireless Power Transmission Emission Element EMIT, such as, but notlimited to, a magnetic coil, or electrical current carrying plate, thatis located substantially nearby an implanted power receiving module,such as is represented by the illustrative example of Housing H, whichis configured to be operatively coupled to Delivery Segment(s) DS.Within Housing H, may be a power supply, light source, and controller,such that the controller activates the light source by controllingcurrent thereto. Alternately, the power receiving module may be locatedat the applicator (not shown), especially when the Applicator isconfigured to contain a Light Source.

An electrical synapse is a mechanical and electrically conductive linkbetween two abutting neurons that is formed at a narrow gap between thepre- and postsynaptic neurons known as a gap junction. At gap junctions,such cells approach within about 3.5 nm of each other, a much shorterdistance than the 20 to 40 nm distance that separates cells at achemical synapse. In many systems, electrical synapse systems co-existwith chemical synapses.

Compared to chemical synapses, electrical synapses conduct nerveimpulses faster, but unlike chemical synapses they do not have gain (thesignal in the postsynaptic neuron is the same or smaller than that ofthe originating neuron). Electrical synapses are often found in neuralsystems that require the fastest possible response, such as defensivereflexes and in cases where a concerted behavior of a subpopulation ofcells is required (such as in propagation of calcium waves inastrocytes, etc.). An important characteristic of electrical synapses isthat most of the time, they are bidirectional, i.e. they allow impulsetransmission in either direction. However, some gap junctions do allowfor communication in only one direction.

Normally, current carried by ions could travel in either directionthrough this type of synapse. However, sometimes the junctions arerectifying synapses, containing voltage-dependent gates that open inresponse to a depolarization and prevent current from traveling in oneof the two directions. Some channels may also close in response toincreased calcium (Ca²⁺) or hydrogen (H+) ion concentration so as not tospread damage from one cell to another.

Certain embodiments of the present invention relate to systems, methodsand apparatuses that provide for optogenetic control of synapticrectification in order to offer improved control for both optogeneticand electrical nerve stimulation.

Nerve stimulation, such as electrical stimulation (“e-stim”), may causebidirectional impulses in a neuron, which may be characterized asantidromic and/or orthodromic stimulation. That is, an action potentialmay trigger pulses that propagate in both directions along a neuron.However, the coordinated use of optogenetic inhibition in combinationwith stimulation may allow only the intended signal to propagate beyondthe target location by suppression or cancellation of the errant signalusing optogenetic inhibition. This may be achieved in multiple waysusing what we will term “multi-applicator devices” or “multi-zonedevices”. The function and characteristics of the individual elementsutilized in such devices were defined earlier.

In a first embodiment, a multi-applicator device is configured toutilize separate applicators Ax for each interaction zone Zx along thetarget nerve N, as is shown in FIG. 39A. One example is the useoptogenetic applicators on both ends (A1, A3) and an electricalstimulation device (A2) in the middle. This example was chosen torepresent a generic situation wherein the desired signal direction maybe on either side of the excitatory electrode. The allowed signaldirection may be chosen by the selective application of optogeneticinhibition from the applicator on the opposite side of the centralApplicator A2. In this non-limiting example, the Errant Impulse EI is onthe RHS of the stimulation cuff A2, traveling to the right, as indicatedby arrow DIR-EI, and passing through the portion f the target covered byA3 and the Desired Impulse DI is on the LHS of A2, travelling to theleft, as indicated by arrow DIR-DI, and, passing through the portion fthe target covered by A1. Activation of A3 may serve to disallowtransmission of EI via optogenetic inhibition of the signal, suppressingit. Similarly, activation of A1 instead of A3 would serve to suppressthe transmission of the Desired Impulse DI and allow the Errant ImpulseEI to propagate. Therefore, bi-directionality is maintained in thistriple applicator configuration, making it a flexible configuration forImpulse direction control. Such flexibility may not always be clinicallyrequired, and simpler designs may be used, as is explained in subsequentparagraphs. This inhibition/suppression signal may accompany or precedethe electrical stimulation, as dictated by the specific kinetics of thetherapeutic target. Each optical applicator may also be made such thatit is capable of providing both optogenetic excitation and inhibition byutilizing two spectrally distinct light sources to activate theirrespective opsins in the target. In this embodiment, each applicator,Ax, is served by its own Delivery Segment, DSx. These Delivery Segments,DS1, DS2, and DS3 serve as conduits for light and/or electricity, asdictated by the type of applicator present. As previously described, theDelivery Segment(s) connect(s) to a Housing containing the electricaland/or electro-optical components required to provide for power supply,processing, feedback, telemetry, etc. Alternately, Applicator A2 may bean optogenetic applicator and either Applicators A1 or A3 may be used tosuppress the errant signal direction.

Alternately, as mentioned above, only a pair of applicators may berequired when the therapy dictates that only a single direction isrequired. Referring to the embodiment of FIG. 39B, the directionality ofthe Desired Impulse DI and Errant Impulse EI described above ismaintained. However, Applicator A3 is absent because the directionalityof the Desired Impulse DI is considered to be fixed as leftward, andApplicator A2 is used for optogenetic suppression of the Errant ImpulseEI, as previously described.

Alternately, referring to the embodiment of FIG. 39C, a singleapplicator may be used, wherein the electrical and optical activationzones Z1, Z2, and Z3 are spatially separated, but still contained withina single applicator A.

Furthermore, the combined electrical stimulation and optical stimulationdescribed herein may also be used for intraoperative tests of inhibitionin which an electrical stimulation is delivered and inhibited by theapplication of light to confirm proper functioning of the implant andoptogenetic inhibition. This may be performed using the applicators andsystem previously described for testing during the surgical procedure,or afterwards, depending upon medical constraints and/or idiosyncrasiesof the patient and/or condition under treatment. The combination of amultiple-applicator, or multiple-zone applicator, or multipleapplicators, may also define which individual optical source elementswithin said applicator or applicators may be the most efficacious and/orefficient means by which to inhibit nerve function. That is, an e-stimdevice may be used as a system diagnostic tool to test the effects ofdifferent emitters and/or applicators within a multiple emitter, ordistributed emitter, system by suppressing, or attempting to suppress,the induced stimulation via optogenetic inhibition using an emitter, ora set of emitters and ascertaining, or measuring, the patient, ortarget, response(s) to see the optimal combination for use. That optimalcombination may then be used as input to configure the system via thetelemetric link to the housing via the external controller/programmer.Alternately, the optimal pulsing characteristics of a single emitter, orset of emitters, may be likewise ascertained and deployed to theimplanted system.

In one embodiment, a system may be configured such that both theinhibitory and excitatory probes and/or applicators are both opticalprobes used to illuminate cells containing light-activatable ionchannels that reside within a target tissue. In this configuration, thecells may be modified using optogenetic techniques, such as has beendescribed elsewhere herein, especially with regard to therapy forcardiac hypertension.

One further embodiment of such a system may be to attach an opticalapplicator, or applicators, on the Vagus nerve to send ascendingstimulatory signals to the brain, while suppressing the descendingsignals by placing the excitatory applicator proximal to the CNS and theinhibitory applicator distal to the excitatory applicator. Theexcitatory applicator may, for example, supply illumination in the rangeof 10-100 mW/mm² of nominally 450±50 nm light to the surface of thenerve bundle to activate cation channels in the cell membrane of thetarget cells within the Vagus nerve while the inhibitory applicatorsupplies illumination in the range of 10-100 mW/mm² of nominally 590±50nm light to activate Cl⁻ ion pumps in the cell membrane of the targetcells to suppress errant descending signals from reaching the PNS.

In an alternate embodiment, the inhibitory probe may be activated priorto the excitatory probe to bias the nerve to suppress errant signals.For example, activating the inhibitory probe at least 5 ms prior to theexcitatory probe allows time for the Cl− pumps to have cycled at leastonce for an opsin such as eNpHR3.0, thus potentially allowing for a morerobust errant signal inhibition. Other opsins have different timeconstants, as described elsewhere herein, and subsequently differentpre-excitation activation times.

Alternately, a system may be configured such that only either theinhibitory or excitatory probes and/or applicators are optical probesused to illuminate cells containing light-activatable ion channels thatreside within a target tissue while other probe is an electrical probe.In the case of the stimulation applicator being an electrical probe,typical neurostimulation parameters, such as those described in U.S.patent application Ser. Nos. 13/707,376 and 13/114,686, which areexpressly incorporated herein by reference, may be used. The operationof a stimulation probe, including alternative embodiments of suitableoutput circuitry for performing the same function of generatingstimulation pulses of a prescribed amplitude and width, is described inU.S. Pat. Nos. 6,516,227 and 6,993,384, which are expressly incorporatedherein by reference. By way of non-limiting example, parameters fordriving an electrical neuroinhibition probe, such as those described inU.S. patent application Ser. No. 12/360,680, which is expresslyincorporated herein by reference, may be used. When the neuroinhibitionis accomplished using an electrical probe, the device may be operated ina mode that is called a “high frequency depolarization block”. By way ofnon-limiting example, for details regarding the parameters for driving ahigh frequency depolarization block electrical probe reference can bemade to Kilgore K L and Bhadra N, High Frequency Mammalian NerveConduction Block: Simulations and Experiments, Engineering in Medicineand Biology Society, 2006. EMBS '06. 28th Annual InternationalConference of the IEEE, pp. 4971-4974, which is expressly incorporatedherein by reference.

In further embodiments, sensors may be used to ascertain the amount oferrant signal suppression in a closed-loop manner to adjust theinhibitory system parameters. An example of such a system is shown inFIG. 39B or 39C, where a sensor SEN is located passed the inhibitionprobe ascertain the degree of errant nerve signal suppression. SensorSEN may be configured to measure the nerve signal by using an ENG probe,for example. It alternately be a therapeutic sensor configured tomonitor a physical therapeutic outcome directly, or indirectly. Such atherapeutic sensor may be, by way of non-limiting example, an ENG probe,an EMG probe, a pressure transducer, a chemical sensor, an EKG sensor,or a motion sensor. A direct sensor is considered to be one thatmonitors a therapeutic outcome directly, such as the aforementionedexamples of chemical and pressure sensors. An indirect sensor is onethat monitors an effect of the treatment, but not the ultimate result.Such sensors are the aforementioned examples of ENG, EKG, and EMGprobes, as has been described elsewhere herein with respect to therapyfor cardiac hypertension.

Alternately, the therapeutic sensor may be a patient input device thatallows the patient to at least somewhat dictate the optical dosageand/or timing. Such a configuration may be utilized, by way ofnon-limiting example, in cases such as muscle spasticity, where thepatient may control the optical dosage and/or timing to provide whatthey deem to be the requisite level of control for a given situation.

As described herein with regard to probe and/or applicator placement,distal refers to more peripheral placement, and proximal refers to morecentral placement along a nerve. As such, an inhibition probe that islocated distally to an excitation probe may be used to provide ascendingnerve signals while suppressing descending nerve signals. Equivalently,this configuration may be described as an excitation probe that islocated proximally to an inhibition probe. Similarly, an excitationprobe that is located distally to an inhibition probe may be used toprovide descending nerve signals while suppress ascending nerve signals.Equivalently, this configuration may be described as an inhibition probethat is located proximally to an excitation probe. Descending signalstravel in the efferent direction away from the CNS towards the PNS, andvice versa ascending signals travel in the afferent direction.

Excitatory opsins useful in the invention may include red-shifteddepolarizing opsins including, by way of non-limiting examples, C1V1 andC1V1 variants C1V1/E162T and C1V1/E122T/E162T; blue depolarizing opsinsincluding ChR2/L132C and ChR2/T159C and combinations of these with theChETA substitutions E123T and E123A; and SFOs including ChR2/C128T,ChR2/C128A, and ChR2/C128S. These opsins may also be useful forinhibition using a depolarization block strategy. Inhibitory opsinsuseful in the invention may include, by way of non-limiting examples,NpHR, Arch, eNpHR3.0 and eArch3.0. Opsins including trafficking motifsmay be useful. An inhibitory opsin may be selected from those listed inFIG. 62J (FIGS. 62J-1 and 62J-2, by way of non-limiting examples. Astimulatory opsin may be selected from those listed in FIG. 62J (FIGS.62J-1 and 62J-2, by way of non-limiting examples. An opsin may beselected from the group consisting of Opto-β2AR or Opto-α1AR, by way ofnon-limiting examples.

In one embodiment, an optogenetic treatment system may be installed andutilized to control cardiovascular hypertension by selectively andcontrollably modulating the activity of the renal nerve plexus.Referring to FIGS. 40A-40B and 41, certain aspects of the human urinarysystem anatomy are depicted, including the right kidney (40), leftkidney (42; shown in partial sectional view in FIG. 40A), a right ureter(44), the bladder (46), and urethra (48). FIG. 40B illustrates that therenal nerve plexus typically at least partially envelops each of therenal arteries. The right renal plexus (50) cascades down in a weblikefashion around the right renal artery and generally includes a rightrenal ganglion (54); similarly, the left renal plexus (52) generallycascades down in a weblike fashion around the left renal artery andgenerally includes a left renal ganglion (56). FIG. 41 illustrates aclose-up partial anatomical schematic view depicting the renal artery(58) as the main vascular intercoupling between the kidney (42) and theaorta (62). The renal plexus (52) generally resides around the renalartery underneath a thin layer of renal fascia (64).

Primary cardiovascular hypertension (the term “primary” used inreference to high blood pressure not caused by another illness) affectssome 20-25% of the adult population worldwide, and persistently elevatedblood pressure levels have been shown to lead to many harmful clinicalconsequences. Heretofore, methods for treating hypertension haveprimarily been pharmacologic, with common drug prescriptions includingdiuretics, adrenergic receptor antagonists, calcium channel blockers,renin inhibitors, ACE inhibitors, angiotensin II receptor antagonists,aldosterone antagonists, vasodilators, and alpha-2 agonists. For somepatients, treatment with one or more of these drug classes succeeds inbringing their blood pressure into a normal range and therefore reducestheir risk of hypertension's many consequences. Many patients, however,remain unresponsive to these drug therapies. Their hypertension persistsdespite an aggressive drug regimen, and they therefore continue on thepathway toward life-altering strokes, dementia, kidney failure, andheart failure. Among physicians, such patients are said to be sufferingfrom “treatment-resistant hypertension.”

Since around 2008, a device-based treatment paradigm known as “renaldenervation” or “ablative renal denervation” has been available fortreatment-resistant hypertension patients. For example, certain aspectsare described in U.S. Pat. No. 6,978,174, which is incorporated byreference herein in its entirety. Essentially this family of proceduresinvolves utilizing a radiofrequency probe such as an endovascularcatheter placed through the renal artery to ablatively destroy portionsof the renal nerve plexus that are positioned adjacent the renal artery.The reason that this procedure has an impact upon hypertension is thatthe kidney normally receives nerve signals from the renal artery nerveplexus that set in motion a series of processes that raise bloodpressure. Referring to FIG. 42, this series of inter-related bodilyfunctions is known to physicians as the “renin-angiotensin-aldosteronesystem”. Elements with “+” indications near them are indicative of astimulatory signal to the system; elements with “−” indications nearthem are indicative of an inhibitory signal to the system. Blockingnerve impulses in the renal nerve plexus essentially places a stop inportions of this process to prevent it from occurring, thereby blockingone of the body's main methods of raising blood pressure. Specifically,when stretch receptors in the upper half of the heart (the atria) sensethat blood pressure is low, they send nerve impulses down the spinalcord, through five spinal roots (T10-S2), through the renal artery nerveplexus, and into the kidneys, activating the juxtaglomerular apparatus(millions of individual juxtaglomerular apparatuses). Thejuxtaglomerular apparatuses respond to the nerve impulses by secreting ahormone known as renin, as shown in FIG. 42. Renin circulates in thebloodstream and acts as an enzyme that converts a protein produced byliver cells, angiotensinogen, into its mature form, angiotensin I.Angiotensin I also circulates in the bloodstream, and when it makes itsway into the lungs, it encounters angiotensin converting enzyme (ACE)which converts it into angiotensin II. Angiotensin II circulatesthroughout the bloodstream and is recognized by receptors on the cellsmembranes of cells lining small arteries. When angiotensin II makescontact with these small-artery-lining cells, they react by triggering acontraction of the smooth muscle cells around the artery, causing theartery to constrict (become smaller). Hence, angiotensin II is known asa natural “vasoconstrictor.” The effect of constricting millions ofsmall blood vessels at the same time is to reduce the total volume ofthe cardiovascular system, which raises blood pressure. Angiotensin IIalso acts to raise blood pressure in other ways, including causing thekidney to retain more fluid than it otherwise would, and increasing therate at which the heart beats.

Hence, by preventing the signal to secrete renin from reaching the cellsin the kidney that produce renin, the entire system is blocked and thebody is unable to raise blood pressure by its natural approach. Patientsthus treated using renal denervation techniques may have effectivelyreduced their blood pressure and thereby reduced their risk of manyill-health consequences, but they also lack an important ability toraise blood pressure when needed (i.e., by permanently ablating ordestroying portions of the subject nerve plexus, this functionality isgone—for better or worse—and these patients are at risk for the reverseproblem: hypotension, or blood pressure that is too low). Hypotensioncan result in fainting, ischemic strokes, and an inability to exercise.Further, as with almost any kind of ablative treatment, the destructionof tissue can involve destruction of tissue that was intended to not bedamaged in the procedure (radiofrequency ablation in a wet,close-quarters environment generally is not hyper-specific). So therenal denervation procedure that has been in use since 2008 may solveone problem (hypertension) but create other problems due to its lack ofspecificity and permanence. Thus there is a need for a configurationthat can reversibly block the renin-angiotensin-aldolsterone axis: onethat can be turned on or off, be adjusted along a range of effect, andone that has specificity. This challenge may be addressed in a novel andunprecedented manner using optogenetic techniques.

Referring ahead to FIG. 45, in one embodiment, a treatment configurationmay comprise preoperative diagnostics and analysis, such as a renalcirculation and/or anatomy study using fluoroscopy, radiography,ultrasound, laparoscopy, or other techniques to understand thevasculature and other structures in detail (74). A polynucleotideencoding a light-responsive opsin protein to be expressed in the neuronsof the renal plexus may be injected (76), after which a waiting time forexpression of the proteins may ensue (78). In an embodiment, opsinslisted herein may be useful in optogenetic methods for treatinghypertension.

A light delivery interface or applicator configuration may then beinstalled (80) and illumination may be delivered through the applicatorto cause inhibition of hypertension through the renal plexus in aspecific and controllable manner (82). As described above, injection maytake many forms, including injection directly into the parenchyma of thekidney (element 60 of FIG. 41) from a transcutaneous position with asyringe for transfective uptake into the renal plexus, direct injectionusing a syringe from a transcutaneous or laparoscopic platform intovarious branches of the renal plexus or ganglion, topical injection orapplication of a vector solution or gel onto various branches of therenal plexus or ganglion, or injection utilizing a movable housing orcuff with a matrix of needles, as described, for example, in referenceto FIGS. 2A and 2B. Also as described above, the illuminationconfiguration, delivery system, and main system housing may take severalforms.

Referring ahead to FIG. 48, in one embodiment, for example, the housing(H) comprises control circuitry and a power supply; the delivery system(DS) comprises an electrical lead to pass power and monitoring signalsas the lead operatively couples the housing (H) to the applicator (A);the applicator (A) preferably comprises a cuff style applicator, akin tothose described in reference to FIGS. 2A-2B but with an illuminatingsubstrate or akin to those described in reference to FIGS. 21A-21C.Alternatively a configuration such as those described in reference toFIGS. 10A-10B may be utilized.

Generally the opsin configuration will be selected to facilitatecontrollable inhibitory neuromodulation of the associated renal nerveplexus in response to light application through the applicator. Thus inone embodiment an inhibitory opsin such as NpHR, eNpHR 3.0, ARCH 3.0, orArchT, or Mac 3.0 may be utilized. In another embodiment, an inhibitoryparadigm may be accomplished by utilizing a stimulatory opsin in ahyperactivation paradigm, as described above. Suitable stimulatoryopsins for hyperactivation inhibition may include ChR2, VChR1, certainStep Function Opsins (ChR2 variants, SFO), ChR2/L132C (CatCH),excitatory opsins listed herein, or a red-shifted C1V1 variant (e.g.,C1V1), the latter of which may assist with illumination penetrationthrough fibrous tissues which may tend to creep in or encapsulate theapplicator (A) relative to the targeted neuroanatomy of the renalplexus. In another embodiment, an SSFO may be utilized. An SFO or anSSFO is differentiated in that it may have a time domain effect for aprolonged period of minutes to hours, which may assist in the downstreamtherapy in terms of saving battery life (i.e., one light pulse may get alonger-lasting physiological result, resulting in less overall lightapplication through the applicator A). As described above, preferablythe associated genetic material is delivered via viral transfection inassociation with injection paradigm, as described above. An inhibitoryopsin may be selected from those listed in FIG. 62J (FIGS. 62J-1 and62J-2, by way of non-limiting examples. A stimulatory opsin may beselected from those listed in FIG. 62J (FIGS. 62J-1 and 62J-2, by way ofnon-limiting examples. An opsin may be selected from the groupconsisting of Opto-β2AR or Opto-α1AR, by way of non-limiting examples.

The virus used can be one of a number of available gene deliveryvectors. Consideration of viral type used in the application todelivering opsins to the relevant neuronal sites innervating the kidneytakes into account delivery to the neurons, selectivity to only theseneurons, trafficking of viral cargoes, safety of the approach andability to effectively express the opsin for prolonged periods such thattherapeutic utility can be maintained. One viral type of use may beadeno-associated viruses (AAV), these viruses have an advantage comparedto other potential viruses in that their DNA does not integrate randomlyinto the host cell genome, this being of benefit as it avoids thepotential of oncogenic consequences. There are multiple serotypes of AAVthat may be of utility in this application. AAV1 can be injected intothe renal parenchyma or the artery itself where it will be taken up intothe nerve terminals present therein; the virus will subsequently beretrogradely transported to the neuronal cell bodies such that thesingle stranded DNA that is delivered to the host cell nucleus will beconverted to episomal concatamers that will be maintained for the lifeof the neuron. Other viral serotypes may be used in this application;the exact preference being combination of ability to be retrogradelytransported to the neuronal cell body, cellular tropism and low level ofimmunogenicity. AAV2 has commonly been used, AAV6 displays lowerimmunogenicity while AAV1, 6, 8 and 9 show high levels of retrogradetransport. The optimal AAV serotype could be determined by one skilledin the art by testing each available serotype and analyzing expressionin neuronal cell bodies and axons.

Other viruses may also be used to deliver the transgene of interest;these include adenovirus, lentiviruses and pseudorabies or rabies virus.These viruses have an added advantage that they can be pseudotyped byreplacing their envelope proteins with those of other viruses or withchimeric envelope proteins that can direct tropism to specific cellularpopulations. By use of pseudotyped viruses that can specificallytransduce the neurons innervating the kidney, a greater specificity canbe obtained which is of benefit, avoiding the expression of opsins inother cells such as the smooth muscle of the renal artery, which wouldotherwise be responsive to illumination and may influence the desiredphysiological function. Specificity may also be achieved by usingcell-type-specific promoters to control the expression of the opsin,even if multiple cell types have been transduced with virus. Promoterswhich allow ubiquitous expression, with little differentiation betweencell types such as the cytomegalovirus (CMV) promoter may be used inthis therapeutic application. However greater selectivity may beachieved using a promoter that directs expression specifically to theneurons innervating the kidney. Examples of such promoters include, butare not limited to, human synapsin and neuronal specific enolase. Forthe promoter chosen there may be as restricted an expression as isnecessary to prevent off target effects, along with sufficientexpression of the opsin such that functional levels may be attained inthe neuron. For specific neuronal cell types, a variety of promoters maybe utilized. E.g. for motor neurons, promoter domains derived fromchicken beta actin (CBA), the transcription factor Hb9, the “survival ofmotor neuron” (SMN1), methyl-CpG-binding protein-2 (MeCP2) and promotersof the transcription factors Pax6, Nkx6.1, Olig2, and Mnr2 may beutilized successfully. For sensory neurons, the latency-associatedpromoter 2 (LAP2), neuron-specific enolase (NSE) may be used. Otherpromoters as listed herein may also be useful.

Additional functionality in the constructs used in this application canbe achieved by the use of specific trafficking and targeting sequencesin the construct. As opsins are only functional when expressed on theplasma membrane of neurons addition of signal sequences that willpromote the trafficking of opsin through the ER, Golgi apparatus andspecifically to the plasma membrane can allow the most efficientexpression. Furthermore, sequences can be incorporated that direct theopsin to specific compartments such as the cell body, axon or dendriteswhich can further increase the presence of opsin in the desiredlocation.

Referring to FIG. 43, the renal fascia (64) and underlying renal arteryand renal nerve plexus may be reached using laparoscopic procedures, asshown in the laparoscopic camera view (70) which shows two smallgrasping/cutting tools extending into the subject anatomy through one ormore relatively small transcutaneous port wounds. Referring to FIG. 44,a cuff-like applicator (A) is installed about the periphery of the renalartery (58) immediately adjacent portions of the renal plexus (52) tofacilitate illumination, as directed by the intercoupled (DS)implantable system housing (H), which preferably is implanted within thepatient as well, as shown, for example, in FIG. 48, wherein the housing(H) resides near the pelvis of the patient. The embodiment of FIG. 48also shows an external programmer/communicator (94) which may bewirelessly connected (i.e., using inductive techniques) to the housing(H) for programming, exchanging data, or inductive battery charging. Theembodiment of FIG. 48 also features an implantable endovascular pressuresensor (90), such as those available from Fraunhofer-Gesellschaft underthe tradename “Hyper-IMS”, which may be connected to the controllerwithin the housing (H) via an electrical lead (88) to facilitateclosed-loop hypertension control (i.e., blood pressure may be monitoredusing the sensor 90 and controlled using the optogenetic control system,H, DS, A), as described in FIG. 49, wherein a sensor may be installed(96) and utilized for closed-loop control of blood pressure (98).

Alternately, a system may be configured to utilize one or more wirelesspower transfer inductors/receivers that are implanted within the body ofa patient that are configured to supply power to the implantable powersupply.

There are a variety of different modalities of inductive coupling andwireless power transfer. For example, there is non-radiative resonantcoupling, such as is available from Witricity, or the more conventionalinductive (near-field) coupling seen in many consumer devices. All areconsidered within the scope of the present invention. The proposedinductive receiver may be implanted into a patient for a long period oftime. Thus, the mechanical flexibility of the inductors may need to besimilar to that of human skin or tissue. Polyimide that is known to bebiocompatible was used for a flexible substrate.

By way of non-limiting example, a planar spiral inductor may befabricated using flexible printed circuit board (FPCB) technologies intoa flexible implantable device. There are many kinds of a planar inductorcoils including, but not limited to; hoop, spiral, meander, and closedconfigurations. In order to concentrate a magnetic flux and fieldbetween two inductors, the permeability of the core material is the mostimportant parameter. As permeability increases, more magnetic flux andfield are concentrated between two inductors. Ferrite has highpermeability, but is not compatible with microfabrication technologies,such as evaporation and electroplating. However, electrodepositiontechniques may be employed for many alloys that have a highpermeability. In particular, Ni (81%) and Fe (19%) composition filmscombine maximum permeability, minimum coercive force, minimum anisotropyfield, and maximum mechanical hardness. An exemplary inductor fabricatedusing such NiFe material may be configured to include 200 μm width traceline width, 1001m width trace line space, and have 40 turns, for aresultant self-inductance of about 25 pH in a device comprising aflexible 24 mm square that may be implanted within the tissue of apatient. The power rate is directly proportional to the self-inductance.

The radio-frequency protection guidelines (RFPG) in many countries suchas Japan and the USA recommend the limits of current for contact hazarddue to an ungrounded metallic object under the electromagnetic field inthe frequency range from 10 kHz to 15 MHz. Power transmission generallyrequires a carrier frequency no higher than tens of MHz for effectivepenetration into the subcutaneous tissue.

In certain embodiments of the present invention, an implanted powersupply may take the form of, or otherwise incorporate, a rechargeablemicro-battery, and/or capacitor, and/or super-capacitor to storesufficient electrical energy to operate the light source and/or othercircuitry within or associated with the implant when used along with anexternal wireless power transfer device. Exemplary microbatteries, suchas the Rechargeable NiMH button cells available from VARTA, are withinthe scope of the present invention. Supercapacitors are also known aselectrochemical capacitors.

FIG. 46 illustrates an embodiment wherein the opsin genetic material andapplicator are installed at the same time (84) to minimize the number ofprocedures to the patient.

FIG. 47 illustrates an embodiment wherein the opsin genetic material andapplicator are installed at the same time by virtue of an applicatorwhich also functions as an injection means (86), as described, forexample, in reference to FIGS. 2A and 2B above.

Referring to FIG. 50, anatomical variation generally may be accountedfor in procedures such as those described herein, and therefore, it maybe desirable in a case wherein a patient has an extra renal artery (100)to have three applicators placed at each of the renal arteries tocontrol the associated portions of the renal plexus. An inhibitory opsinprotein may be selected from the group consisting of, by way ofnon-limiting examples: NpHR, eNpHR 1.0, eNpHR 2.0, eNpHR 3.0, Mac, Mac3.0, Arch, Arch3.0, and ArchT. An inhibitory opsin may be selected fromthose listed in FIG. 62J (FIGS. 62J-1 and 62J-2, by way of non-limitingexamples. A stimulatory opsin protein may be selected from the groupconsisting of, by way of non-limiting examples: ChR2, C1V1-E122T,C1V1-E162T, C1V1-E122T/E162T, CatCh, VChR1-SFO, and ChR2-SFO. Astimulatory opsin may be selected from those listed in FIG. 62J (FIGS.62J-1 and 62J-2, by way of non-limiting examples. An opsin may beselected from the group consisting of Opto-β2AR or Opto-α1AR, by way ofnon-limiting examples. The light source may be controlled to deliver apulse duration between about 0.1 and about 20 milliseconds, a duty cyclebetween about 0.1 and 100 percent, and a surface irradiance of betweenabout 5 milliwatts per square millimeter to about 200 milliwatts persquare millimeter.

FIGS. 88A and 88B show an alternate embodiment of the present invention,where a Trocar and Cannula may be used to deploy an at least partiallyimplantable system for optogenetic control of the renal nerve plexus forthe control of cardiac hypertension. Trocar TROCAR may be used to createa tunnel through tissue between surgical access points that maycorrespond to the approximate intended deployment locations of elementsof the present invention, such as applicators and housings. CannulaCANNULA may be inserted into the tissue of the patient along with, orafter the insertion of the trocar. The trocar may be removed followinginsertion and placement of the cannula to provide an open lumen for theintroduction of system elements. The open lumen of cannula CANNULA maythen provide a means to locate delivery segment DS along the routebetween a housing and an applicator. The ends of delivery segment DS maybe covered by end caps ENDC. End caps ENDC may be further configured tocomprise radio-opaque markings ROPM to enhance the visibility of thedevice under fluoroscopic imaging and/or guidance. End Caps ENDC mayprovide a watertight seal to ensure that the optical surfaces of theDelivery Segment DS, or other system component being implanted, are notdegraded. The cannula may be removed subsequent to the implantation ofdelivery segment DS. Subsequently, delivery segment DS may be connectedto an applicator that is disposed to the target tissue and/or a housing,as have been described elsewhere herein. In a further embodiment, theEnd Caps ENDC, or the Delivery Segment DS itself may be configured toalso include a temporary Tissue Fixation elements AFx, such as, but notlimited to; hook, tines, and barbs, that allow the implanted device toreside securely in its location while awaiting further manipulation andconnection to the remainder of the system.

FIG. 89 illustrates an alternate embodiment, similar to that of FIGS.88A&B, further configured to utilize a barbed Tissue Fixation Element AFthat is affixed to End Cap ENDC. Tissue Fixation Element AF may be abarbed, such that it will remain substantially in place after insertionalong with Cannula CANNULA, shown in this example as a hypodermic needlewith sharp End SHARP being the leading end of the device as it isinserted into a tissue of a patient. The barbed feature(s) of TissueFixation Element AF insert into tissue, substantially disallowingDelivery Segment DS to be removed. In a still further embodiment, TissueFixation Element AF may be made responsive to an actuator, such as atrigger mechanism (not shown) such that it is only in the configurationto affirmatively remain substantially in place after insertion whenactivated, thus providing for the ability to be relocated more easilyduring the initial implantation, and utilized in conjunction with aforward motion of Delivery Segment DS to free the end from the tissue ithas captured. Delivery Segment DS may be substantially inside the hollowcentral lumen of Cannula CANNULA, or substantially slightly forward ofit, as is shown in the illustrative embodiment. As used herein, cannulaalso refers to an elongate member, or delivery conduit. The elongatedelivery conduit may be a cannula. The elongate delivery conduit may bea catheter. The catheter may be a steerable catheter. The steerablecatheter may be a robotically steerable catheter, configured to haveelectromechanical elements induce steering into the elongate deliveryconduit in response to commands made by an operator with an electronicmaster input device that is operatively coupled to the electromechanicalelements. The surgical method of implantation further may compriseremoving the elongate delivery conduit, leaving the delivery segment inplace between the first anatomical location and the second anatomicallocation.

An alternate embodiment of the invention may comprise the use of a SFOand/or a SSFO opsin in the cells of the target tissue to affect neuralinhibition of the renal plexus for the treatment of cardiachypertension, such a system may comprise a 2-color illumination systemin order to activate and then subsequently deactivate the lightsensitive protein. As is described elsewhere herein, the step functionopsins may be activated using blue or green light, such as a nominally450 nm LED or laser light source, and may be deactivated using a yellowor red light, such as a nominally 600 nm LED of laser light source. Thetemporal coordination of these colors may be made to produce ahyperstimulation (depolarization) block condition by pulsing the firstlight source for activation to create an activation pulse of a durationbetween 0.1 and 10 ms, then pulsing the second light source fordeactivation to create a deactivation pulse of a duration between 0.1 to10 ms at a time between 1 and 100 ms after the completion of theactivation pulse from the first light source. Alternately, certaininhibitory opsins, such as, but not limited to, NpHR and Arch, may besimilarly deactivated using blue light.

It is understood that systems for renal nerve inhibition may beconfigured from combinations of any of the applicators,controllers/housings, delivery segments, and other system elementsdescribed, and utilize therapeutic parameters defined herein. By way ofnon-limiting example, a system comprising a nominally 590 nm LED lightsource may be operatively coupled to a waveguide delivery segment,comprised of a bundle of 37 100 μm diameter optical fibers, via ahermetic optical feedthrough to transmit light from within animplantable housing, and controlled by a controller therein, to anaxially rolled slab-type applicator, comprised of multiple outputcouplers and a fitted with a reflective sleeve, that may be disposed onor about the exterior of the renal artery to illuminate cells containingan NpHR opsin within the target tissue with a pulse duration of between0.1-10 ms, a duty cycle of between 20-50%, and an irradiance of between5-20 mW/mm² at the surface of the renal artery.

FIG. 102 shows an alternate exemplary embodiment of a system for thetreatment of cardiac hypertension via optogenetic inhibition of therenal nerve plexus, comprising elements, such as is described in moredetail with respect to FIGS. 10-26, 31-34, 37, 40-50, 64-69, 73A-75, and87-89. Applicator A, a rolled slab-type applicator that is 10 mm wideand 40 mm long when unrolled, such as is described in more detail withrespect to FIGS. 18 and 21-23 is deployed about Renal Artery 58, whichcontains Renal Plexus 52, of Kidney 42, such as is described in moredetail with respect to FIGS. 40-50. Applicator A further comprises InnerSurface IS and Outer Surface OS, such as is described in more detailwith respect to FIG. 21B, wherein Outer Surface OS may be at least apartially reflecting surface configured to recycle remitted light backinto Target Tissue N (which, in this example, is Renal Plexus 52).Applicator A further comprises Sensor SEN1, such as is described in moredetail with respect to FIGS. 24 and 69. Light is delivered to ApplicatorA via Delivery Segments DS, such as is described in more detail withrespect to FIGS. 10-21. Connector C is configured to operatively couplelight from Delivery Segments DS to Applicator A, such as is described inmore detail with respect to FIGS. 10A and 64-68. Delivery Segments DSfurther comprise Undulations U, such as is described in more detail withrespect to FIGS. 17B and 82-82. Delivery Segments DS are furtherconfigured to comprise Signal Wires SW between Sensor SEN1 and theController CONT of Housing H. As such, Connector C is further configuredto provide the electrical connection, as well. Delivery Segments DS areoperatively coupled to Housing H via Optical Feedthrough OFT, such as isdescribed in more detail with respect to FIGS. 73A-75. Light is providedto Delivery Segments DS from Light Sources LS1 and LS2 within Housing Hafter being combined with Beam Combiner BC, such as is described in moredetail with respect to FIG. 16. Light Sources LS1 and LS2 may beconfigured to LEDs, and/or lasers that provide spectrally differentoutput to activate and/or deactivate the opsins resident within TargetTissue 52, such as is described in more detail elsewhere herein. TheController CONT shown within Housing H is a simplification, for clarity,of that described in more detail with respect to FIGS. 32-34. Externalclinician programmer module and/or a patient programmer module C/P maycommunicate with Controller CONT via Telemetry module TM via Antenna ANTvia Communications Link CL, such as is described in more detail withrespect to FIGS. 31-32 and 39. Power Supply PS, not shown for clarity,may be wirelessly recharged using External Charger EC, such as isdescribed in more detail with respect to FIGS. 31-34. Furthermore,External Charger EC may be configured to reside within a Mounting DeviceMOUNTING DEVICE, such as is described in more detail with respect toFIG. 87. Mounting Device MOUNTING DEVICE may be a pant, as is especiallywell configured for this exemplary embodiment. External Charger EC, aswell as External clinician programmer module and/or a patient programmermodule C/P and Mounting Device MOUNTING DEVICE may be located within theextracorporeal space ESP, while the rest of the system is implanted andmay be located within the intracorporeal space ISP, such as is describedin more detail with respect to FIGS. 31 and 70. The system may furthercomprise an implanted in-situ blood pressure sensor 90, resident withinthe Femoral Artery, such as is described in more detail with respect toFIG. 48. Implantable endovascular pressure sensor 90, such as thoseavailable from Fraunhofer-Gesellschaft under the tradename “Hyper-IMS”,may be connected to the controller within the housing (H) via anelectrical lead (88) to facilitate closed-loop hypertension control.Electrical Lead 88 may be connected to the Controller CONT of Housing Hvia an Electrical Feedthrough EFT, such as, by way of non-limitingexample, The SYGNUS® Implantable Contact System from Bal-SEAL.

In certain scenarios wherein light sensitivity of opsin genetic materialmay be of paramount importance, it may be desirable to focus less onwavelength (as discussed above, certain “red-shifted” opsins may beadvantageous due to the greater permeability of the associated radiationwavelengths through materials such as tissue structures) and more on atradeoff that has been shown between response time and light sensitivity(or absorption cross-section). In other words, optimal opsin selectionin many applications may be a function of system kinetics and lightsensitivity. Referring to the plot (252) of FIG. 62A, for example,electrophysiology dose for a 50% response (or “EPD50”; lower EPD50 meansmore light-sensitive) is plotted versus temporal precision (“τ_(off)”,which represents the time constant with which an opsin deactivates afterthe illumination has been discontinued). These data are from Mattis etal, Nat Methods 2011, Dec. 10; 9(2): 159-172, which is incorporated byreference herein in its entirety, and illustrates the aforementionedtradeoff. In addition to EPD50 and τ_(off), other important factorsplaying into opsin selection optimization may include exposure density(“H-thresh”) and photocurrent levels. H-thresh may be assessed bydetermining the EPD50 dose for an opsin; the longer the channel createdby the opsin requires to “reset”, the longer the associated membranewill remain polarized, and thus will block further depolarization. Thefollowing table features a few exemplary opsins with characteristicscompared.

Pentration Depth Tau- Lambda [normalized Peak SS Peak EPD50 off Peak toPhotocurrent Photocurrent Potential Opsin [mW/mm2] [ms] [nm] 475 nm][nA] [nA] [mV] C1V1t 0.3 75 540 1.67 1.5 1 30 C1V1tt 0.4 50 540 1.67 1.10.6 32 Catch 0.3 60 475 1.00 1.25 1 38 VChR1 0.1 100 550 1.80

Thus, from an opsin protein selection perspective, the combination oflow exposure density (H-thresh), long photorecovery time (τ_(off)), andhigh photocurrent results in an opsin well-suited for applications thatdo not require ultra-temporal precision. As described above, a furtherconsideration remains the optical penetration depth of the light orradiation responsible for activating the opsin. Tissue is a turbidmedium, and predominantly attenuates the power density of light by Mie(elements of similar size to the wavelength of light) and Rayleigh(elements of smaller size than the wavelength of light) scatteringeffects. Both effects are inversely proportional to the wavelength, i.e.shorter wavelength is scattered more than a longer wavelength. Thus, alonger opsin excitation wavelength is preferred, but not required, forconfigurations where there is tissue interposed between the illuminationsource and the target. A balance may be made between the ultimateirradiance (optical power density and distribution) at the target tissuecontaining the opsin and the response of the opsin itself. Thepenetration depth in tissue (assuming a simple lambda⁻⁴scatteringdependence) is listed in the table above. Considering all theabovementioned parameters, both C1V1(E162T) and VChR1 may be desirablechoices in many clinical scenarios, due to combination of low exposurethreshold, long photorecovery time, and optical penetration depth. FIGS.62B-62C and 62E-62I feature further plots (254, 256, 260, 262, 264, 266,268, respectively) containing data from the aforementioned incorporatedMattis et al 2011 reference, demonstrating the interplay/relationshipsof various parameters of candidate opsins. FIG. 62D features a plot(258) similar to that shown in FIG. 4B, which contains data from Yizharet al, Neuron. 2011 July; 72:9-34, which is incorporated by referenceherein in its entirety. The table (270) of FIG. 62J (FIGS. 62J-1 and62J-2 features data from the aforementioned incorporated Yizhar et alNeuron 2011 reference, in addition to Wang et al, 2009, Journal ofBiological Chemistry, 284: 5625-5696; Gradinaru et al, 2010, Cell:141:1-12; Wen et al., PLoS One. 2010; 5(9):e12893; Lin et al, Biophys J.2009; 96(5):1803-14; Lin et al., Nat Neurosci. 2013 16(10):1499-1508,all of which are incorporated by reference herein in their entirety.

Amino acid sequences of exemplary opsins, as well as of exemplary signalpeptides, signal sequences, ER export sequences, and a traffickingsequence, are shown in FIGS. 51A-61M-7. Information on exemplary opsins,signal peptides, signal sequences, ER export sequences, and traffickingsequences is also in published US patent applications 20130019325 and2011011217, published PCT application WO/2013/126521, Yizhar et al,Nature. 2011; 477(7363):171-8; Zhang F, et al., Cell. 2011;147(7):1446-57; Mattis et al., Nat Methods. 2011; 9(2):159-72; and Fennoet al., Annu Rev Neurosci. 2011; 34:389-412, Prakash et al., 2012 NatureMethods 9(12):1171-1179; as well as in the GenBank records cited inFIGS. 51A-61M-7, all of which are incorporated by reference.

Referring to FIGS. 90-101, various configurations for conductingprocedures featuring technologies such as those described above areillustrated. Referring to FIG. 90, for example, one embodiment isillustrated for controlling hypertension of a patient via optogeneticintervention, wherein along with patient analysis and diagnostics(1030), such as blood pressure characterization, echocardiography,performance stress testing, and/or blood chemistry testing, a tissuestructure such as the renal nerve pelvis may be targeted forintervention (1032). The targeted tissue structure may be geneticallymodified to encode a light sensitive protein (such as by such as byviral mediated gene delivery, electroporation, ultrasound, hydrodynamicdelivery, or introduction of naked DNA by direct injection or ascomplemented by additional facilitators such as cationic lipids orpolymers; 1034) and an optical applicator may be implanted and coupledto a tissue structure in a configuration allowing the optical applicatorto deliver photons to at least one branch of the targeted tissuestructure, such as the renal nerve plexus, which has been geneticallymodified to have the light sensitive protein (1036). An implantablelight source and implantable power supply may be implanted and coupledto one or more tissue structures to provide stability, the implantablelight source being configured to deliver photons as an input to theoptical applicator when the implantable light source is drawing powerfrom the implantable power supply (1038). In implantable sensor may bedeployed within the body of the patient and configured to provide anoutput signal that is correlated with the blood pressure of the patient(1040). An implantable controller may be implanted and coupled to atleast one tissue structure; the implantable controller may be configuredto cause the implantable light source to direct enough illumination tothe light sensitive protein through the implantable light source andimplantable light applicator to at least partially inhibit actionpotential transmission within the at least one branch of the renal nerveplexus based at least in part upon the output signal received from theimplantable sensor (1042). Thus a closed-loop hypertension controlparadigm may be executed utilizing light sensitive proteins.

Referring to FIG. 91, an embodiment somewhat similar to that of FIG. 90is illustrated, with the exception that the embodiment of FIG. 91features an open-loop paradigm without a blood pressure sensor, suchthat along with patient analysis and diagnostics (1030), identificationof a targeted tissue structure such as a branch of the renal nerveplexus (1032), genetic modification of the targeted tissue structure toencode a light sensitive opsin protein (1034), implantation of anoptical applicator (1036) and implantable light source (1038), animplantable controller is implanted to be operatively coupled to theimplantable light source and configured to cause the implantable lightsource to direct enough illumination to the light sensitive proteinthrough the implantable light source and implantable optical applicatorto at least partially inhibit action potential transmission within theat least one branch of the renal nerve plexus (1044). The controller maybe configured to induce or cause chronic stimulation (i.e., over a longperiod of time, somewhat akin to a cardiac pacemaker pacingfunctionality, but in this example the pacing may be stimulating aninhibitory-opsin-encoding nerve to prevent the creation/propagation ofaction potentials) to prevent the renal plexus from elevating thepatient's blood pressure.

Referring to FIG. 92, a closed loop illuminance configuration isdepicted wherein along with patient analysis and diagnostics (1030) andidentification of a targeted tissue structure, such as one or moreneurons or nerves (1048), the targeted tissue structure may begenetically modified to encode a light sensitive opsin protein (1034),and a implantable optical applicator may be provided to deliver light tothe targeted tissue structure after implantation in a location adjacentto the targeted tissue structure (1050). The implantable opticalapplicator may be operatively coupled to a light source, a controller, apower supply, and an implantable illuminance sensor such that thecontroller causes the power supply to allow current to flow to the lightsource to cause an emission of photons to the implantable light actuatorbased at least in part upon an output signal from the implantableilluminance sensor, wherein the implantable illuminance sensor ispositioned such that it captures at least a portion of the photonsdirected toward the targeted tissue structure by the implantable lightapplicator (1052).

Referring to FIG. 93, a configuration is illustrated wherein directionalcontrol of an action potential may be achieved in a nerve comprisinglight sensitive protein. As shown in FIG. 93, along with patientanalysis and diagnostics (1030) and identification of a targeted tissuestructure, such as one or more neurons or nerves (1048), the targetedtissue structure may be genetically modified to encode a light sensitiveopsin protein (1034), and an implantable optical applicator may beprovided and configured to engage the targeted nerve and to deliverlight to the targeted nerve, the implantable optical applicatoroperatively coupled to a light source configured to deliver photons tothe implantable optical applicator which may be emitted by theimplantable optical applicator into the targeted nerve to cause amembrane polarization change in the targeted nerve (1054). Animplantable electrical stimulation applicator, such as an “e-stim” orelectrical stimulation electrode, configured to engage and stimulate thetargeted nerve may be provided, the implantable electrical stimulationapplicator operatively coupled to a power source configured to deliverelectrons to the implantable electrical stimulation applicator which maybe emitted by the implantable electrical stimulation applicator into thetargeted nerve to cause a membrane polarization change in the targetednerve (1056). A controller may be operatively coupled to the lightsource and the power source, and configured to cause both current toflow to the implantable electrical stimulation applicator and photons tobe directed to the implantable optical applicator, such that an actionpotential is created which propagates in a first desired direction alongthe nerve, and which does not substantially propagate in a reversedirection along the nerve (1058).

Referring to FIG. 94, another configuration is illustrated whereindirectional control of an action potential may be achieved in a nervecomprising light sensitive protein. As shown in FIG. 94, along withpatient analysis and diagnostics (1030) and identification of a targetedtissue structure, such as one or more neurons or nerves (1048), thetargeted tissue structure may be genetically modified to encode twodifferent light sensitive opsin proteins, such as by viral mediated genedelivery, electroporation, ultrasound, hydrodynamic delivery, orintroduction of naked DNA by direct injection or as complemented byadditional facilitators such as cationic lipids or polymers, asdiscussed above (1060). A first implantable optical applicator may beprovided and configured to engage the targeted nerve and to deliverlight to the targeted nerve, the first implantable optical applicatoroperatively coupled to a first light source configured to deliverphotons to the first implantable optical applicator which may be emittedby the first implantable optical applicator into the targeted nerve tocause a membrane polarization change in the targeted nerve (1062). Asecond implantable optical applicator may be provided and configured toengage the targeted nerve and to deliver light to the targeted nerve,the second implantable optical applicator operatively coupled to asecond light source configured to deliver photons to the secondimplantable optical applicator which may be emitted by the secondimplantable optical applicator into the targeted nerve to cause amembrane polarization change in the targeted nerve (1064). A controllermay be operatively coupled to the first light source and second lightsource, the controller configured to cause photons to be directed toeach of the first and second implantable optical applicators, such that,depending upon the particular types of opsin proteins selected anddelivered to the pertinent nerve, an action potential is created whichpropagates in a first desired direction along the nerve, and which doesnot substantially propagate in a reverse direction along the nerve(1068).

Referring to FIG. 95, a method for installing a system for stimulating atargeted tissue structure comprising light sensitive protein isillustrated, wherein certain components are pre-coupled. As shown inFIG. 95, along with along with patient analysis and diagnostics (1030)and identification of a targeted tissue structure, such as one or moreneurons or nerves (1048), the targeted tissue structure may begenetically modified to encode a light sensitive opsin protein (1034),and an implantable optical applicator may be provided and configured tobe permanently operatively coupled to at least a portion of the targetedtissue structure, an implantable light source, an implantable powersupply, an implantable controller, wherein the implantable opticalapplicator, implantable light source, implantable power supply, andimplantable controller are provided in a pre-coupled andhermetically-sealed configuration and designed to be installed togetherthrough a common surgical access port (1070). A common surgical accessport to a first anatomical location may be created at which theimplantable power supply and implantable controller are to be implanted(1072). An elongate delivery conduit defining a working lumentherethrough may be inserted along an insertion pathway to a secondanatomical location without creating further surgical access for theinsertion pathway, the second anatomical location being adjacent thetargeted tissue structure (1074). The implantable optical applicator maybe inserted through the elongate delivery conduit to implant theimplantable optical applicator at the second anatomical locationoperatively coupled to at least a portion of the targeted tissuestructure, such that a least a portion of photons delivered through theimplantable optical applicator will reach the targeted tissue structure(1076).

Referring to FIG. 96, a method for installing a system for stimulating atargeted tissue structure comprising light sensitive protein isillustrated, wherein certain components are coupled in-situ. As shown inFIG. 96, along with patient analysis and diagnostics (1030) andidentification of a targeted tissue structure, such as one or moreneurons or nerves (1048), the targeted tissue structure may begenetically modified to encode a light sensitive opsin protein (1034),and an implantable optical applicator may be provided and configured tobe permanently operatively coupled to at least a portion of the targetedtissue structure, an implantable light source, an implantable powersupply, an implantable controller (1078). A common surgical access portmay be created to a first anatomical location at which the implantablepower supply and implantable controller are to be implanted (1080). Anelongate delivery conduit defining a working lumen therethrough may beinserted along an insertion pathway to a second anatomical locationwithout creating further surgical access for the insertion pathway, thesecond anatomical location being adjacent the targeted tissue structure(1082). A second surgical access port to the second anatomical locationmay be created (1084), and the implantable optical applicator may beimplanted at the second anatomical location through the second surgicalaccess port (1086). The implantable power supply (such as animplantable/sealed battery) and implantable controller (such as amicrocontroller, microprocessor, application specific integratedcircuit, or field programmable gate array, for example) may be implantedat the first anatomical location through the common access port (1088)and a delivery segment may be inserted through the elongate deliveryconduit to operatively couple the implantable optical applicator withthe implantable power supply and implantable controller (1090).

Referring to FIG. 97, a method for installing a system for stimulating atargeted tissue structure comprising light sensitive protein isillustrated, wherein certain components may be coupled in-situ andcertain components may be pre-coupled. As shown in FIG. 97, along withpatient analysis and diagnostics (1030) and identification of a targetedtissue structure, such as one or more neurons or nerves (1048), thetargeted tissue structure may be genetically modified to encode a lightsensitive opsin protein (1034), and an implantable optical applicatormay be provided and configured to be permanently operatively coupled toat least a portion of the targeted tissue structure, an implantablelight source, an implantable power supply, an implantable controller,wherein the implantable power supply and implantable controller areprovided in a pre-coupled and hermetically-sealed configuration anddesigned to be installed together through a common surgical access port(1086). A common surgical access port may be created to a firstanatomical location at which the implantable power supply andimplantable controller are to be implanted (1088). An elongate deliveryconduit defining a working lumen therethrough may be inserted along aninsertion pathway to a second anatomical location without creatingfurther surgical access for the insertion pathway, the second anatomicallocation being adjacent the targeted tissue structure (1090). Theimplantable optical applicator may be inserted through the elongatedelivery conduit to implant the implantable optical applicator at thesecond anatomical location operatively coupled to at least a portion ofthe targeted tissue structure, such that a least a portion of photonsdelivered through the implantable optical applicator will reach thetargeted tissue structure (1092).

Referring to FIG. 98, a method for illuminating a targeted tissuestructure of a patient is depicted. As shown in FIG. 98, along withpatient analysis and diagnostics (1030) and identification of a targetedtissue structure, such as one or more neurons or nerves (1048), thetargeted tissue structure may be genetically modified to encode a lightsensitive opsin protein (1034), and an implantable optical applicatormay be provided and configured to engage the outer surface of thetargeted tissue structure and to deliver light directly to the targetedtissue structure (1094). The implantable optical applicator may beimplanted such that it engages the targeted tissue structure (1096) andthe implantable optical applicator may be operatively coupled to a lightsource (1098). The targeted tissue structure may be illuminated throughthe implantable light applicator using photons transferred to theimplantable light applicator from the light source (1100).

Referring to FIG. 99, a method for stimulating a tissue structurecomprising light sensitive protein is illustrated. As shown in FIG. 99,along with patient analysis and diagnostics (1030) and identification ofa targeted tissue structure, such as one or more neurons or nerves to bestimulated using illumination at a first subcutaneous location (1102),the targeted tissue structure may be genetically modified to encode alight sensitive opsin protein (1034), and an implantable lightconductor, such as a light pipe or waveguide, may be provided andconfigured to be permanently coupled between the first subcutaneouslocation and a second location selected such that extracorporeal photonsdirected toward the second location will be transmitted, at least inpart, through the implantable light conductor to the targeted tissuestructure (1104).

Referring to FIG. 100, a method for illuminating, from within theintrathecal space of a spine of a patient, a nerve root pair that hasbeen genetically modified to comprise light sensitive protein isillustrated. As shown in FIG. 100, along with patient analysis anddiagnostics (1030) and identification of a targeted tissue structure,such as one or more neurons, nerve roots, or nerves to be stimulatedusing illumination at a first subcutaneous location (1106), the targetedtissue structure may be genetically modified to encode a light sensitiveopsin protein (1034), and an implantable optical applicator may besurgically delivered in a compressed state through a small surgicalaccess created between calcified structures of the spine to theintrathecal space (1108). The implantable optical applicator may beexpanded inside of the intrathecal space to an expanded state, whereinthe implantable optical applicator comprises a first lighting segmentand a second lighting segment, the first lighting segment configured toilluminate a first nerve root of a selected nerve root pair while thesecond lighting segment is configured to illuminate a second nerve rootfrom the selected nerve root pair (1110).

Referring to FIG. 101, a configuration for controllably injecting atargeted tissue structure is illustrated. As shown in FIG. 101, alongwith patient analysis and diagnostics (1030) and selection of a targetedtissue structure to be injected (1112; such as one or more neurons,nerve roots, or nerves to be stimulated using illumination after beinggenetically modified, such as by viral mediated gene delivery using aninjection, as described above), an elongate flexible injection memberbiased to assume an arcuate geometric configuration when unloaded may beprovided, the injection member comprising an array of injection needlesand being configured to assume a substantially straight geometricconfiguration based upon an input from an operator manipulating a remoteactuator that is operatively coupled to the elongate flexible injectionmember (1114). The flexible injection member may be caused to assume thesubstantially straight configuration based upon an input from theoperator (1116) and may be navigated into a position relative to thetargeted tissue structure such that upon release of the input, theflexible injection member will be biased to return to the arcuategeometric configuration, and in reconfiguring toward the arcuateconfiguration, portions of the elongate flexible member will advance aplurality of injection needles into the targeted tissue structure(1118). The input may be released to advance the plurality of theinjection needles into the targeted tissue structure (1120) and thetargeted tissue structure may be controllably injected using theplurality of the needles advanced into the targeted tissue structure(1122).

In some embodiments, the light-responsive protein can comprise an aminoacid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to the sequence shown in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:49. In anembodiment, the light-responsive protein can comprise an amino acidsequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to a polypeptide encoded by SEQ ID NO:50.

An “individual” can be a mammal, including a human. Mammals include, butare not limited to, farm animals, sport animals, pets, primates, miceand rats. Individuals also include companion animals including, but notlimited to, dogs and cats. In one aspect, an individual is a human. Inanother aspect, an individual is a non-human animal.

As used herein, “depolarization-induced synaptic depletion” occurs whencontinuous depolarization of a neural cell plasma membrane prevents theneural cell from sustaining high frequency action on efferent targetsdue to depletion of terminal vesicular stores of neurotransmitters.

Amino acid substitutions in a native protein sequence may be“conservative” or “non-conservative” and such substituted amino acidresidues may or may not be one encoded by the genetic code. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a chemicallysimilar side chain (i.e., replacing an amino acid possessing a basicside chain with another amino acid with a basic side chain). A“non-conservative amino acid substitution” is one in which the aminoacid residue is replaced with an amino acid residue having a chemicallydifferent side chain (i.e., replacing an amino acid having a basic sidechain with an amino acid having an aromatic side chain).

The standard twenty amino acid “alphabet” is divided into chemicalfamilies based on chemical properties of their side chains. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and side chains having aromatic groups (e.g.,tyrosine, phenylalanine, tryptophan, histidine).

As used herein, an “effective dosage” or “effective amount” of drug,compound, or pharmaceutical composition is an amount sufficient toeffect beneficial or desired results. For prophylactic use, beneficialor desired results include results such as eliminating or reducing therisk, lessening the severity, or delaying the onset of the disease,including biochemical, histological and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. For therapeutic use,beneficial or desired results include clinical results such asdecreasing one or more symptoms resulting from the disease, increasingthe quality of life of those suffering from the disease, decreasing thedose of other medications required to treat the disease, enhancingeffect of another medication such as via targeting, delaying theprogression of the disease, and/or prolonging survival. An effectivedosage can be administered in one or more administrations. For purposesof this invention, an effective dosage of drug, compound, orpharmaceutical composition is an amount sufficient to accomplishprophylactic or therapeutic treatment either directly or indirectly. Asis understood in the clinical context, an effective dosage of a drug,compound, or pharmaceutical composition may or may not be achieved inconjunction with another drug, compound, or pharmaceutical composition.Thus, an “effective dosage” may be considered in the context ofadministering one or more therapeutic agents, and a single agent may beconsidered to be given in an effective amount if, in conjunction withone or more other agents, a desirable result may be or is achieved.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to, one or more of the following: decreasing symptomsresulting from the disease, increasing the quality of life of thosesuffering from the disease, decreasing the dose of other medicationsrequired to treat the disease, delaying the progression of the disease,and/or prolonging survival of individuals.

Light-Responsive Opsin Proteins

Provided herein are optogenetic-based methods for selectivelyhyperpolarizing or depolarizing neurons.

Optogenetics refers to the combination of genetic and optical methodsused to control specific events in targeted cells of living tissue, evenwithin freely moving mammals and other animals, with the temporalprecision (millisecond-timescale) needed to keep pace with functioningintact biological systems. Optogenetics requires the introduction offast light-responsive channel or pump proteins to the plasma membranesof target neuronal cells that allow temporally precise manipulation ofneuronal membrane potential while maintaining cell-type resolutionthrough the use of specific targeting mechanisms. Any microbial opsinthat can be used to promote neural cell membrane hyperpolarization ordepolarization in response to light may be used. For example, theHalorhodopsin family of light-responsive chloride pumps (e.g., NpHR,NpHR2.0, NpHR3.0, NpHR3.1) and the GtR3 proton pump can be used topromote neural cell membrane hyperpolarization in response to light. Asanother example, eARCH (a proton pump) or ArchT can be used to promoteneural cell membrane hyperpolarization in response to light.Additionally, members of the Channelrhodopsin family of light-responsivecation channel proteins (e.g., ChR2, SFOs, SSFOs, C1V1s) can be used topromote neural cell membrane depolarization or depolarization-inducedsynaptic depletion in response to a light stimulus.

Enhanced Intracellular Transport Amino Acid Motifs

The present disclosure provides for the modification of light-responsiveopsin proteins expressed in a cell by the addition of one or more aminoacid sequence motifs which enhance transport to the plasma membranes ofmammalian cells. Light-responsive opsin proteins having componentsderived from evolutionarily simpler organisms may not be expressed ortolerated by mammalian cells or may exhibit impaired subcellularlocalization when expressed at high levels in mammalian cells.

Consequently, in some embodiments, the light-responsive opsin proteinsexpressed in a cell can be fused to one or more amino acid sequencemotifs selected from the group consisting of a signal peptide, anendoplasmic reticulum (ER) export signal, a membrane trafficking signal,and/or an N-terminal golgi export signal. The one or more amino acidsequence motifs which enhance light-responsive protein transport to theplasma membranes of mammalian cells can be fused to the N-terminus, theC-terminus, or to both the N- and C-terminal ends of thelight-responsive protein. Optionally, the light-responsive protein andthe one or more amino acid sequence motifs may be separated by a linker.In some embodiments, the light-responsive protein can be modified by theaddition of a trafficking signal (ts) which enhances transport of theprotein to the cell plasma membrane. In some embodiments, thetrafficking signal can be derived from the amino acid sequence of thehuman inward rectifier potassium channel Kir2.1. In other embodiments,the trafficking signal can comprise the amino acid sequenceKSRITSEGEYIPLDQIDINV (SEQ ID NO:37).

Trafficking sequences that are suitable for use can comprise an aminoacid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%, amino acid sequence identity to an amino acid sequence such atrafficking sequence of human inward rectifier potassium channel Kir2.1(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:37)).

A trafficking sequence can have a length of from about 10 amino acids toabout 50 amino acids, e.g., from about 10 amino acids to about 20 aminoacids, from about 20 amino acids to about 30 amino acids, from about 30amino acids to about 40 amino acids, or from about 40 amino acids toabout 50 amino acids.

Signal sequences that are suitable for use can comprise an amino acidsequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, amino acid sequence identity to an amino acid sequence such as oneof the following:

1) the signal peptide of hChR2 (e.g., MDYGGALSAVGRELLFVTNPVVVNGS (SEQ IDNO:38))

2) the 32 subunit signal peptide of the neuronal nicotinic acetylcholinereceptor (e.g., MAGHSNSMALFSFSLLWLCSGVLGTEF (SEQ ID NO:39));

3) a nicotinic acetylcholine receptor signal sequence (e.g.,MGLRALMLWLLAAAGLVRESLQG (SEQ ID NO:40)); and

4) a nicotinic acetylcholine receptor signal sequence (e.g.,MRGTPLLLVVSLFSLLQD (SEQ ID NO:41)).

A signal sequence can have a length of from about 10 amino acids toabout 50 amino acids, e.g., from about 10 amino acids to about 20 aminoacids, from about 20 amino acids to about 30 amino acids, from about 30amino acids to about 40 amino acids, or from about 40 amino acids toabout 50 amino acids.

Endoplasmic reticulum (ER) export sequences that are suitable for use ina modified opsin of the present disclosure include, e.g., VXXSL (where Xis any amino acid) [SEQ ID NO:42](e.g., VKESL (SEQ ID NO:43); VLGSL (SEQID NO:44); etc.); NANSFCYENEVALTSK (SEQ ID NO:45); FXYENE (SEQ ID NO:46)(where X is any amino acid), e.g., FCYENEV (SEQ ID NO:47); and the like.An ER export sequence can have a length of from about 5 amino acids toabout 25 amino acids, e.g., from about 5 amino acids to about 10 aminoacids, from about 10 amino acids to about 15 amino acids, from about 15amino acids to about 20 amino acids, or from about 20 amino acids toabout 25 amino acids.

Additional protein motifs which can enhance light-responsive proteintransport to the plasma membrane of a cell are described in U.S. patentapplication Ser. No. 12/041,628, which is incorporated herein byreference in its entirety. In some embodiments, the signal peptidesequence in the protein can be deleted or substituted with a signalpeptide sequence from a different protein.

Light-Responsive Chloride Pumps

In some aspects of the methods provided herein, one or more members ofthe Halorhodopsin family of light-responsive chloride pumps areexpressed on the plasma membranes of neural cells.

In some aspects, said one or more light-responsive chloride pumpproteins expressed on the plasma membranes of the nerve cells describedabove can be derived from Natronomonas pharaonis. In some embodiments,the light-responsive chloride pump proteins can be responsive to amberlight as well as red light and can mediate a hyperpolarizing current inthe nerve cell when the light-responsive chloride pump proteins areilluminated with amber or red light. The wavelength of light which canactivate the light-responsive chloride pumps can be between about 580and 630 nm. In some embodiments, the light can be at a wavelength ofabout 589 nm or the light can have a wavelength greater than about 630nm (e.g. less than about 740 nm). In another embodiment, the light has awavelength of around 630 nm. In some embodiments, the light-responsivechloride pump protein can hyperpolarize a neural membrane for at leastabout 90 minutes when exposed to a continuous pulse of light. In someembodiments, the light-responsive chloride pump protein can comprise anamino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO:32.Additionally, the light-responsive chloride pump protein can comprisesubstitutions, deletions, and/or insertions introduced into a nativeamino acid sequence to increase or decrease sensitivity to light,increase or decrease sensitivity to particular wavelengths of light,and/or increase or decrease the ability of the light-responsive proteinto regulate the polarization state of the plasma membrane of the cell.In some embodiments, the light-responsive chloride pump protein containsone or more conservative amino acid substitutions. In some embodiments,the light-responsive protein contains one or more non-conservative aminoacid substitutions.

The light-responsive protein comprising substitutions, deletions, and/orinsertions introduced into the native amino acid sequence suitablyretains the ability to hyperpolarize the plasma membrane of a neuronalcell in response to light.

Additionally, in other aspects, the light-responsive chloride pumpprotein can comprise a core amino acid sequence at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence shown in SEQ ID NO: 32 and an endoplasmic reticulum (ER) exportsignal. This ER export signal can be fused to the C-terminus of the coreamino acid sequence or can be fused to the N-terminus of the core aminoacid sequence. In some embodiments, the ER export signal is linked tothe core amino acid sequence by a linker. The linker can comprise any ofabout 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275,300, 400, or 500 amino acids in length. The linker may further comprisea fluorescent protein, for example, but not limited to, a yellowfluorescent protein, a red fluorescent protein, a green fluorescentprotein, or a cyan fluorescent protein. In some embodiments, the ERexport signal can comprise the amino acid sequence FXYENE (SEQ IDNO:46), where X can be any amino acid. In another embodiment, the ERexport signal can comprise the amino acid sequence VXXSL, where X can beany amino acid [SEQ ID NO:42]. In some embodiments, the ER export signalcan comprise the amino acid sequence FCYENEV (SEQ ID NO:47).

Endoplasmic reticulum (ER) export sequences that are suitable for use ina modified opsin of the present disclosure include, e.g., VXXSL (where Xis any amino acid) [SEQ ID NO:42](e.g., VKESL (SEQ ID NO:43); VLGSL (SEQID NO:44); etc.); NANSFCYENEVALTSK (SEQ ID NO:45); FXYENE (where X isany amino acid) (SEQ ID NO:46), e.g., FCYENEV (SEQ ID NO:47); and thelike. An ER export sequence can have a length of from about 5 aminoacids to about 25 amino acids, e.g., from about 5 amino acids to about10 amino acids, from about 10 amino acids to about 15 amino acids, fromabout 15 amino acids to about 20 amino acids, or from about 20 aminoacids to about 25 amino acids.

In other aspects, the light-responsive chloride pump proteins providedherein can comprise a light-responsive protein expressed on the cellmembrane, wherein the protein comprises a core amino acid sequence atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence shown in SEQ ID NO: 32 and a traffickingsignal (e.g., which can enhance transport of the light-responsivechloride pump protein to the plasma membrane). The trafficking signalmay be fused to the C-terminus of the core amino acid sequence or may befused to the N-terminus of the core amino acid sequence. In someembodiments, the trafficking signal can be linked to the core amino acidsequence by a linker which can comprise any of about 5, 10, 20, 30, 40,50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 aminoacids in length. The linker may further comprise a fluorescent protein,for example, but not limited to, a yellow fluorescent protein, a redfluorescent protein, a green fluorescent protein, or a cyan fluorescentprotein. In some embodiments, the trafficking signal can be derived fromthe amino acid sequence of the human inward rectifier potassium channelKir2.1. In other embodiments, the trafficking signal can comprise theamino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:37).

In some aspects, the light-responsive chloride pump protein can comprisea core amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ IDNO: 32 and at least one (such as one, two, three, or more) amino acidsequence motifs which enhance transport to the plasma membranes ofmammalian cells selected from the group consisting of an ER exportsignal, a signal peptide, and a membrane trafficking signal. In someembodiments, the light-responsive chloride pump protein comprises anN-terminal signal peptide, a C-terminal ER Export signal, and aC-terminal trafficking signal. In some embodiments, the C-terminal ERExport signal and the C-terminal trafficking signal can be linked by alinker. The linker can comprise any of about 5, 10, 20, 30, 40, 50, 75,100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids inlength. The linker can also further comprise a fluorescent protein, forexample, but not limited to, a yellow fluorescent protein, a redfluorescent protein, a green fluorescent protein, or a cyan fluorescentprotein. In some embodiments the ER Export signal can be moreC-terminally located than the trafficking signal. In other embodimentsthe trafficking signal is more C-terminally located than the ER Exportsignal. In some embodiments, the signal peptide comprises the amino acidsequence MTETLPPVTESAVALQAE (SEQ ID NO:48). In another embodiment, thelight-responsive chloride pump protein comprises an amino acid sequenceat least 95% identical to SEQ ID NO:33.

Moreover, in other aspects, the light-responsive chloride pump proteinscan comprise a core amino acid sequence at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequenceshown in SEQ ID NO: 32, wherein the N-terminal signal peptide of SEQ IDNO:32 is deleted or substituted. In some embodiments, other signalpeptides (such as signal peptides from other opsins) can be used. Thelight-responsive protein can further comprise an ER transport signaland/or a membrane trafficking signal described herein. In someembodiments, the light-responsive chloride pump protein comprises anamino acid sequence at least 95% identical to SEQ ID NO:34.

In some embodiments, the light-responsive opsin protein is a NpHR opsinprotein comprising an amino acid sequence at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% identical to the sequenceshown in SEQ ID NO:32. In some embodiments, the NpHR opsin proteinfurther comprises an endoplasmic reticulum (ER) export signal and/or amembrane trafficking signal. For example, the NpHR opsin proteincomprises an amino acid sequence at least 95% identical to the sequenceshown in SEQ ID NO:32 and an endoplasmic reticulum (ER) export signal.In some embodiments, the amino acid sequence at least 95% identical tothe sequence shown in SEQ ID NO:32 is linked to the ER export signalthrough a linker. In some embodiments, the ER export signal comprisesthe amino acid sequence FXYENE (SEQ ID NO:46), where X can be any aminoacid. In another embodiment, the ER export signal comprises the aminoacid sequence VXXSL, where X can be any amino acid [SEQ ID NO:42]. Insome embodiments, the ER export signal comprises the amino acid sequenceFCYENEV (SEQ ID NO:47). In some embodiments, the NpHR opsin proteincomprises an amino acid sequence at least 95% identical to the sequenceshown in SEQ ID NO:32, an ER export signal, and a membrane traffickingsignal. In other embodiments, the NpHR opsin protein comprises, from theN-terminus to the C-terminus, the amino acid sequence at least 95%identical to the sequence shown in SEQ ID NO:32, the ER export signal,and the membrane trafficking signal. In other embodiments, the NpHRopsin protein comprises, from the N-terminus to the C-terminus, theamino acid sequence at least 95% identical to the sequence shown in SEQID NO:32, the membrane trafficking signal, and the ER export signal. Insome embodiments, the membrane trafficking signal is derived from theamino acid sequence of the human inward rectifier potassium channelKir2.1. In some embodiments, the membrane trafficking signal comprisesthe amino acid sequence K S R I T S E G E Y I P L D Q I D I N V (SEQ IDNO:37). In some embodiments, the membrane trafficking signal is linkedto the amino acid sequence at least 95% identical to the sequence shownin SEQ ID NO:32 by a linker. In some embodiments, the membranetrafficking signal is linked to the ER export signal through a linker.The linker may comprise any of 5, 10, 20, 30, 40, 50, 75, 100, 125, 150,175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. Thelinker may further comprise a fluorescent protein, for example, but notlimited to, a yellow fluorescent protein, a red fluorescent protein, agreen fluorescent protein, or a cyan fluorescent protein. In someembodiments, the light-responsive opsin protein further comprises anN-terminal signal peptide. In some embodiments, the light-responsiveopsin protein comprises the amino acid sequence of SEQ ID NO:33. In someembodiments, the light-responsive opsin protein comprises the amino acidsequence of SEQ ID NO:34.

Also provided herein are polynucleotides encoding any of thelight-responsive chloride ion pump proteins described herein, such as alight-responsive protein comprising a core amino acid sequence at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence shown in SEQ ID NO:32, an ER export signal,and a membrane trafficking signal. In another embodiment, thepolynucleotides comprise a sequence which encodes an amino acid at least95% identical to SEQ ID NO:33 and SEQ ID NO:34. The polynucleotides maybe in an expression vector (such as, but not limited to, a viral vectordescribed herein). The polynucleotides may be used for expression of thelight-responsive chloride ion pump proteins.

Further disclosure related to light-responsive chloride pump proteinscan be found in U.S. Patent Application Publication Nos: 2009/0093403and 2010/0145418 as well as in International Patent Application No:PCT/US2011/028893, the disclosures of each of which are herebyincorporated by reference in their entireties.

Light-Responsive Proton Pumps

In some aspects of the methods provided herein, one or morelight-responsive proton pumps are expressed on the plasma membranes ofthe neural cells.

In some embodiments, the light-responsive proton pump protein can beresponsive to blue light and can be derived from Guillardia theta,wherein the proton pump protein can be capable of mediating ahyperpolarizing current in the cell when the cell is illuminated withblue light. The light can have a wavelength between about 450 and about495 nm or can have a wavelength of about 490 nm. In another embodiment,the light-responsive proton pump protein can comprise an amino acidsequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to the sequence shown in SEQ ID NO:31. Thelight-responsive proton pump protein can additionally comprisesubstitutions, deletions, and/or insertions introduced into a nativeamino acid sequence to increase or decrease sensitivity to light,increase or decrease sensitivity to particular wavelengths of light,and/or increase or decrease the ability of the light-responsive protonpump protein to regulate the polarization state of the plasma membraneof the cell. Additionally, the light-responsive proton pump protein cancontain one or more conservative amino acid substitutions and/or one ormore non-conservative amino acid substitutions. The light-responsiveproton pump protein comprising substitutions, deletions, and/orinsertions introduced into the native amino acid sequence suitablyretains the ability to hyperpolarize the plasma membrane of a neuronalcell in response to light.

In other aspects of the methods disclosed herein, the light-responsiveproton pump protein can comprise a core amino acid sequence at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence shown in SEQ ID NO:31 and at least one (suchas one, two, three, or more) amino acid sequence motifs which enhancetransport to the plasma membranes of mammalian cells selected from thegroup consisting of a signal peptide, an ER export signal, and amembrane trafficking signal. In some embodiments, the light-responsiveproton pump protein comprises an N-terminal signal peptide and aC-terminal ER export signal. In some embodiments, the light-responsiveproton pump protein comprises an N-terminal signal peptide and aC-terminal trafficking signal. In some embodiments, the light-responsiveproton pump protein comprises an N-terminal signal peptide, a C-terminalER Export signal, and a C− terminal trafficking signal. In someembodiments, the light-responsive proton pump protein comprises a C−terminal ER Export signal and a C-terminal trafficking signal. In someembodiments, the C-terminal ER Export signal and the C-terminaltrafficking signal are linked by a linker. The linker can comprise anyof about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250,275, 300, 400, or 500 amino acids in length. The linker may furthercomprise a fluorescent protein, for example, but not limited to, ayellow fluorescent protein, a red fluorescent protein, a greenfluorescent protein, or a cyan fluorescent protein. In some embodimentsthe ER Export signal is more C-terminally located than the traffickingsignal. In some embodiments the trafficking signal is more C-terminallylocated than the ER Export signal.

Also provided herein are isolated polynucleotides encoding any of thelight-responsive proton pump proteins described herein, such as alight-responsive proton pump protein comprising a core amino acidsequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to the sequence shown in SEQ ID NO:31. Alsoprovided herein are expression vectors (such as a viral vector describedherein) comprising a polynucleotide encoding the proteins describedherein, such as a light-responsive proton pump protein comprising a coreamino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO:31.The polynucleotides may be used for expression of the light-responsiveprotein in neural cells.

Further disclosure related to light-responsive proton pump proteins canbe found in International Patent Application No. PCT/US2011/028893, thedisclosure of which is hereby incorporated by reference in its entirety.

In some embodiments, the light-responsive proton pump protein can beresponsive to green or yellow light and can be derived from Halorubrumsodomense or Halorubrum sp. TP009, wherein the proton pump protein canbe capable of mediating a hyperpolarizing current in the cell when thecell is illuminated with green or yellow light. The light can have awavelength between about 560 and about 570 nm or can have a wavelengthof about 566 nm. In another embodiment, the light-responsive proton pumpprotein can comprise an amino acid sequence at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence shown in SEQ ID NO:25 or SEQ ID NO:26. The light-responsiveproton pump protein can additionally comprise substitutions, deletions,and/or insertions introduced into a native amino acid sequence toincrease or decrease sensitivity to light, increase or decreasesensitivity to particular wavelengths of light, and/or increase ordecrease the ability of the light-responsive proton pump protein toregulate the polarization state of the plasma membrane of the cell.Additionally, the light-responsive proton pump protein can contain oneor more conservative amino acid substitutions and/or one or morenon-conservative amino acid substitutions. The light-responsive protonpump protein comprising substitutions, deletions, and/or insertionsintroduced into the native amino acid sequence suitably retains theability to hyperpolarize the plasma membrane of a neuronal cell inresponse to light.

In other aspects of the methods disclosed herein, the light-responsiveproton pump protein can comprise a core amino acid sequence at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence shown in SEQ ID NO:25 or SEQ ID NO:26 and atleast one (such as one, two, three, or more) amino acid sequence motifswhich enhance transport to the plasma membranes of mammalian cellsselected from the group consisting of a signal peptide, an ER exportsignal, and a membrane trafficking signal. In some embodiments, thelight-responsive proton pump protein comprises an N-terminal signalpeptide and a C-terminal ER export signal. In some embodiments, thelight-responsive proton pump protein comprises an N-terminal signalpeptide and a C-terminal trafficking signal. In some embodiments, thelight-responsive proton pump protein comprises an N-terminal signalpeptide, a C-terminal ER Export signal, and a C-terminal traffickingsignal. In some embodiments, the light-responsive proton pump proteincomprises a C-terminal ER Export signal and a C-terminal traffickingsignal. In some embodiments, the C-terminal ER Export signal and theC-terminal trafficking signal are linked by a linker. The linker cancomprise any of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linkermay further comprise a fluorescent protein, for example, but not limitedto, a yellow fluorescent protein, a red fluorescent protein, a greenfluorescent protein, or a cyan fluorescent protein. In some embodimentsthe ER Export signal is more C-terminally located than the traffickingsignal. In some embodiments the trafficking signal is more C-terminallylocated than the ER Export signal.

Also provided herein are isolated polynucleotides encoding any of thelight-responsive proton pump proteins described herein, such as alight-responsive proton pump protein comprising a core amino acidsequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to the sequence shown in SEQ ID NO:25 or SEQ IDNO:26. Also provided herein are expression vectors (such as a viralvector described herein) comprising a polynucleotide encoding theproteins described herein, such as a light-responsive proton pumpprotein comprising a core amino acid sequence at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence shown in SEQ ID NO:25 or SEQ ID NO:26. The polynucleotides maybe used for expression of the light-responsive protein in neural cells.

Light-Responsive Cation Channel Proteins

In some aspects of the methods provided herein, one or morelight-responsive cation channels can be expressed on the plasmamembranes of the neural cells.

In some aspects, the light-responsive cation channel protein can bederived from Chlamydomonas reinhardtii, wherein the cation channelprotein can be capable of mediating a depolarizing current in the cellwhen the cell is illuminated with light. In another embodiment, thelight-responsive cation channel protein can comprise an amino acidsequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to the sequence shown in SEQ ID NO:1. The lightused to activate the light-responsive cation channel protein derivedfrom Chlamydomonas reinhardtii can have a wavelength between about 460and about 495 nm or can have a wavelength of about 480 nm. Additionally,the light can have an intensity of at least about 100 Hz. In someembodiments, activation of the light-responsive cation channel derivedfrom Chlamydomonas reinhardtii with light having an intensity of 100 Hzcan cause depolarization-induced synaptic depletion of the neuronsexpressing the light-responsive cation channel. The light-responsivecation channel protein can additionally comprise substitutions,deletions, and/or insertions introduced into a native amino acidsequence to increase or decrease sensitivity to light, increase ordecrease sensitivity to particular wavelengths of light, and/or increaseor decrease the ability of the light-responsive cation channel proteinto regulate the polarization state of the plasma membrane of the cell.Additionally, the light-responsive cation channel protein can containone or more conservative amino acid substitutions and/or one or morenon-conservative amino acid substitutions. The light-responsive protonpump protein comprising substitutions, deletions, and/or insertionsintroduced into the native amino acid sequence suitably retains theability to depolarize the plasma membrane of a neuronal cell in responseto light.

In some embodiments, the light-responsive cation channel comprises aT159C substitution of the amino acid sequence set forth in SEQ ID NO:1.In some embodiments, the light-responsive cation channel comprises aL132C substitution of the amino acid sequence set forth in SEQ ID NO:1.In some embodiments, the light-responsive cation channel comprises anE123T substitution of the amino acid sequence set forth in SEQ ID NO:1.In some embodiments, the light-responsive cation channel comprises anE123A substitution of the amino acid sequence set forth in SEQ ID NO:1.In some embodiments, the light-responsive cation channel comprises aT159C substitution and an E123T substitution of the amino acid sequenceset forth in SEQ ID NO:1. In some embodiments, the light-responsivecation channel comprises a T159C substitution and an E123A substitutionof the amino acid sequence set forth in SEQ ID NO:1. In someembodiments, the light-responsive cation channel comprises a T159Csubstitution, an L132C substitution, and an E123T substitution of theamino acid sequence set forth in SEQ ID NO:1. In some embodiments, thelight-responsive cation channel comprises a T159C substitution, an L132Csubstitution, and an E123A substitution of the amino acid sequence setforth in SEQ ID NO:1. In some embodiments, the light-responsive cationchannel comprises an L132C substitution and an E123T substitution of theamino acid sequence set forth in SEQ ID NO:1. In some embodiments, thelight-responsive cation channel comprises an L132C substitution and anE123A substitution of the amino acid sequence set forth in SEQ ID NO:1.

Further disclosure related to light-responsive cation channel proteinscan be found in U.S. Patent Application Publication No. 2007/0054319 andInternational Patent Application Publication Nos. WO 2009/131837 and WO2007/024391, the disclosures of each of which are hereby incorporated byreference in their entireties.

Step Function Opsins and Stabilized Step Function Opsins

In other embodiments, the light-responsive cation channel protein can bea step function opsin (SFO) protein or a stabilized step function opsin(SSFO) protein that can have specific amino acid substitutions at keypositions throughout the retinal binding pocket of the protein. In someembodiments, the SFO protein can have a mutation at amino acid residueC128 of SEQ ID NO:1. In other embodiments, the SFO protein has a C128Amutation in SEQ ID NO:1. In other embodiments, the SFO protein has aC128S mutation in SEQ ID NO:1. In another embodiment, the SFO proteinhas a C128T mutation in SEQ ID NO:1. In some embodiments, the SFOprotein can comprise an amino acid sequence at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence shown in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

In some embodiments, the SFO protein can have a mutation at amino acidresidue D156 of SEQ ID NO:1. In some embodiments, the SFO protein cancomprise an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQID NO:5.

In other embodiments, the SSFO protein can have a mutation at both aminoacid residues C128 and D156 of SEQ ID NO:1. In one embodiment, the SSFOprotein has an C128S and a D156A mutation in SEQ ID NO:1. In anotherembodiment, the SSFO protein can comprise an amino acid sequence atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence shown in SEQ ID NO:6. In another embodiment,the SSFO protein can comprise a C128T mutation in SEQ ID NO:1. In someembodiments, the SSFO protein comprises C128T and D156A mutations in SEQID NO:1.

In some embodiments the SFO or SSFO proteins provided herein can becapable of mediating a depolarizing current in the cell when the cell isilluminated with blue light. In other embodiments, the light can have awavelength of about 445 nm. Additionally, the light can have anintensity of about 100 Hz. In some embodiments, activation of the SFO orSSFO protein with light having an intensity of 100 Hz can causedepolarization-induced synaptic depletion of the neurons expressing theSFO or SSFO protein. In some embodiments, each of the disclosed stepfunction opsin and stabilized step function opsin proteins can havespecific properties and characteristics for use in depolarizing themembrane of a neuronal cell in response to light.

Further disclosure related to SFO or SSFO proteins can be found inInternational Patent Application Publication No. WO 2010/056970 and U.S.Provisional Patent Application Nos. 61/410,704 and 61/511,905, thedisclosures of each of which are hereby incorporated by reference intheir entireties.

C1V1 Chimeric Cation Channels

In other embodiments, the light-responsive cation channel protein can bea C1V1 chimeric protein derived from the VChR1 protein of Volvox carteriand the ChR1 protein from Chlamydomonas reinhardti, wherein the proteincomprises the amino acid sequence of VChR1 having at least the first andsecond transmembrane helices replaced by the first and secondtransmembrane helices of ChR1; is responsive to light; and is capable ofmediating a depolarizing current in the cell when the cell isilluminated with light. In some embodiments, the C1V1 protein canfurther comprise a replacement within the intracellular loop domainlocated between the second and third transmembrane helices of thechimeric light responsive protein, wherein at least a portion of theintracellular loop domain is replaced by the corresponding portion fromChR1. In another embodiment, the portion of the intracellular loopdomain of the C1V1 chimeric protein can be replaced with thecorresponding portion from ChR1 extending to amino acid residue A145 ofthe ChR1. In other embodiments, the C1V1 chimeric protein can furthercomprise a replacement within the third transmembrane helix of thechimeric light responsive protein, wherein at least a portion of thethird transmembrane helix is replaced by the corresponding sequence ofChR1. In yet another embodiment, the portion of the intracellular loopdomain of the C1V1 chimeric protein can be replaced with thecorresponding portion from ChR1 extending to amino acid residue W163 ofthe ChR1. In other embodiments, the C1V1 chimeric protein can comprisean amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO:13or SEQ ID NO:49.

In some embodiments, the C1V1 protein can mediate a depolarizing currentin the cell when the cell is illuminated with green light. In otherembodiments, the light can have a wavelength of between about 540 nm toabout 560 nm. In some embodiments, the light can have a wavelength ofabout 542 nm. In some embodiments, the C1V1 chimeric protein is notcapable of mediating a depolarizing current in the cell when the cell isilluminated with violet light. In some embodiments, the chimeric proteinis not capable of mediating a depolarizing current in the cell when thecell is illuminated with light having a wavelength of about 405 nm.Additionally, the light can have an intensity of about 100 Hz. In someembodiments, activation of the C1V1 chimeric protein with light havingan intensity of 100 Hz can cause depolarization-induced synapticdepletion of the neurons expressing the C1V1 chimeric protein. In someembodiments, the disclosed C1V1 chimeric protein can have specificproperties and characteristics for use in depolarizing the membrane of aneuronal cell in response to light.

C1V1 Chimeric Mutant Variants

In some aspects, the present disclosure provides polypeptides comprisingsubstituted or mutated amino acid sequences, wherein the mutantpolypeptide retains the characteristic light-activatable nature of theprecursor C1V1 chimeric polypeptide but may also possess alteredproperties in some specific aspects. For example, the mutantlight-responsive C1V1 chimeric proteins described herein can exhibit anincreased level of expression both within an animal cell or on theanimal cell plasma membrane; an altered responsiveness when exposed todifferent wavelengths of light, particularly red light; and/or acombination of traits whereby the chimeric C1V1 polypeptide possess theproperties of low desensitization, fast deactivation, low violet-lightactivation for minimal cross-activation with other light-responsivecation channels, and/or strong expression in animal cells.

Accordingly, provided herein are C1V1 chimeric light-responsive opsinproteins that can have specific amino acid substitutions at keypositions throughout the retinal binding pocket of the VChR1 portion ofthe chimeric polypeptide. In some embodiments, the C1V1 protein can havea mutation at amino acid residue E122 of SEQ ID NO:13 or SEQ ID NO:49.In some embodiments, the C1V1 protein can have a mutation at amino acidresidue E162 of SEQ ID NO:13 or SEQ ID NO:49. In other embodiments, theC1V1 protein can have a mutation at both amino acid residues E162 andE122 of SEQ ID NO:13 or SEQ ID NO:49. In other embodiments, the C1V1protein can comprise an amino acid sequence at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence shown in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, or SEQ ID NO:19. In some embodiments, each of thedisclosed mutant C1V1 chimeric proteins can have specific properties andcharacteristics for use in depolarizing the membrane of an animal cellin response to light.

In some aspects, the C1V1-E122 mutant chimeric protein is capable ofmediating a depolarizing current in the cell when the cell isilluminated with light. In some embodiments the light can be greenlight. In other embodiments, the light can have a wavelength of betweenabout 540 nm to about 560 nm. In some embodiments, the light can have awavelength of about 546 nm. In other embodiments, the C1V1-E122 mutantchimeric protein can mediate a depolarizing current in the cell when thecell is illuminated with red light. In some embodiments, the red lightcan have a wavelength of about 630 nm. In some embodiments, theC1V1-E122 mutant chimeric protein does not mediate a depolarizingcurrent in the cell when the cell is illuminated with violet light. Insome embodiments, the chimeric protein does not mediate a depolarizingcurrent in the cell when the cell is illuminated with light having awavelength of about 405 nm. Additionally, the light can have anintensity of about 100 Hz. In some embodiments, activation of theC1V1-E122 mutant chimeric protein with light having an intensity of 100Hz can cause depolarization-induced synaptic depletion of the neuronsexpressing the C1V1-E122 mutant chimeric protein. In some embodiments,the disclosed C1V1-E122 mutant chimeric protein can have specificproperties and characteristics for use in depolarizing the membrane of aneuronal cell in response to light.

In other aspects, the C1V1-E162 mutant chimeric protein is capable ofmediating a depolarizing current in the cell when the cell isilluminated with light. In some embodiments the light can be greenlight. In other embodiments, the light can have a wavelength of betweenabout 535 nm to about 540 nm. In some embodiments, the light can have awavelength of about 542 nm. In other embodiments, the light can have awavelength of about 530 nm. In some embodiments, the C1V1-E162 mutantchimeric protein does not mediate a depolarizing current in the cellwhen the cell is illuminated with violet light. In some embodiments, thechimeric protein does not mediate a depolarizing current in the cellwhen the cell is illuminated with light having a wavelength of about 405nm. Additionally, the light can have an intensity of about 100 Hz. Insome embodiments, activation of the C1V1-E162 mutant chimeric proteinwith light having an intensity of 100 Hz can causedepolarization-induced synaptic depletion of the neurons expressing theC1V1-E162 mutant chimeric protein. In some embodiments, the disclosedC1V1-E162 mutant chimeric protein can have specific properties andcharacteristics for use in depolarizing the membrane of a neuronal cellin response to light.

In yet other aspects, the C1V1-E122/E162 mutant chimeric protein iscapable of mediating a depolarizing current in the cell when the cell isilluminated with light. In some embodiments the light can be greenlight. In other embodiments, the light can have a wavelength of betweenabout 540 nm to about 560 nm. In some embodiments, the light can have awavelength of about 546 nm. In some embodiments, the C1V1-E122/E162mutant chimeric protein does not mediate a depolarizing current in thecell when the cell is illuminated with violet light. In someembodiments, the chimeric protein does not mediate a depolarizingcurrent in the cell when the cell is illuminated with light having awavelength of about 405 nm. In some embodiments, the C1V1-E122/E162mutant chimeric protein can exhibit less activation when exposed toviolet light relative to C1V1 chimeric proteins lacking mutations atE122/E162 or relative to other light-responsive cation channel proteins.Additionally, the light can have an intensity of about 100 Hz. In someembodiments, activation of the C1V1-E122/E162 mutant chimeric proteinwith light having an intensity of 100 Hz can causedepolarization-induced synaptic depletion of the neurons expressing theC1V1-E122/E162 mutant chimeric protein. In some embodiments, thedisclosed C1V1-E122/E162 mutant chimeric protein can have specificproperties and characteristics for use in depolarizing the membrane of aneuronal cell in response to light.

Further disclosure related to C1V1 chimeric cation channels as well asmutant variants of the same can be found in U.S. Provisional PatentApplication Nos. 61/410,736, 61/410,744, and 61/511,912, the disclosuresof each of which are hereby incorporated by reference in theirentireties.

Champ

In some embodiments, the light-responsive protein is a chimeric proteincomprising Arch-TS-p2A-ASIC 2a-TS-EYFP-ER-2 (Champ). Champ comprises anArch domain and an Acid-sensing ion channel (ASIC)-2a domain. Lightactivation of Champ activates a proton pump (Arch domain) that activatesthe ASIC-2a proton-activated cation channel (ASIC-2a domain). Apolynucleotide encoding Champ is shown in SEQ ID NO:50.

Polynucleotides

The disclosure also provides polynucleotides comprising a nucleotidesequence encoding a light-responsive protein described herein. In someembodiments, the polynucleotide comprises an expression cassette. Insome embodiments, the polynucleotide is a vector comprising theabove-described nucleic acid. In some embodiments, the nucleic acidencoding a light-responsive protein of the disclosure is operably linkedto a promoter. Promoters are well known in the art. Any promoter thatfunctions in the host cell can be used for expression of thelight-responsive opsin proteins and/or any variant thereof of thepresent disclosure. In one embodiment, the promoter used to driveexpression of the light-responsive opsin proteins can be a promoter thatis specific to motor neurons. In other embodiments, the promoter iscapable of driving expression of the light-responsive opsin proteins inneurons of both the sympathetic and/or the parasympathetic nervoussystems. Initiation control regions or promoters, which are useful todrive expression of the light-responsive opsin proteins or variantthereof in a specific animal cell are numerous and familiar to thoseskilled in the art. Virtually any promoter capable of driving thesenucleic acids can be used. Examples of motor neuron-specific genes canbe found, for example, in Kudo, et al., Human Mol. Genetics, 2010,19(16): 3233-3253, the contents of which are hereby incorporated byreference in their entirety. In some embodiments, the promoter used todrive expression of the light-responsive protein can be the Thy1promoter, which is capable of driving robust expression of transgenes inneurons of both the central and peripheral nervous systems (See, e.g.,Llewellyn, et al., 2010, Nat. Med., 16(10):1161-1166). In otherembodiments, the promoter used to drive expression of thelight-responsive protein can be the EFla promoter, a cytomegalovirus(CMV) promoter, the CAG promoter, a synapsin-I promoter (e.g., a humansynapsin-I promoter), a human synuclein 1 promoter, a human Thy1promoter, a calcium/calmodulin-dependent kinase II alpha (CAMKIIα)promoter, or any other promoter capable of driving expression of thelight-responsive opsin proteins in the peripheral neurons of mammals.

Also provided herein are vectors comprising a nucleotide sequenceencoding a light-responsive protein or any variant thereof describedherein. The vectors that can be administered according to the presentinvention also include vectors comprising a nucleotide sequence whichencodes an RNA (e.g., an mRNA) that when transcribed from thepolynucleotides of the vector will result in the accumulation oflight-responsive opsin proteins on the plasma membranes of target animalcells. Vectors which may be used, include, without limitation,lentiviral, HSV, adenoviral, and adeno-associated viral (AAV) vectors.Lentiviruses include, but are not limited to HIV-1, HIV-2, SIV, FIV andEIAV. Lentiviruses may be pseudotyped with the envelope proteins ofother viruses, including, but not limited to VSV, rabies, Mo-MLV,baculovirus and Ebola. Such vectors may be prepared using standardmethods in the art.

In some embodiments, the vector is a recombinant AAV vector. AAV vectorsare DNA viruses of relatively small size that can integrate, in a stableand site-specific manner, into the genome of the cells that they infect.They are able to infect a wide spectrum of cells without inducing anyeffects on cellular growth, morphology or differentiation, and they donot appear to be involved in human pathologies. The AAV genome has beencloned, sequenced and characterized. It encompasses approximately 4700bases and contains an inverted terminal repeat (ITR) region ofapproximately 145 bases at each end, which serves as an origin ofreplication for the virus. The remainder of the genome is divided intotwo essential regions that carry the encapsidation functions: theleft-hand part of the genome, that contains the rep gene involved inviral replication and expression of the viral genes; and the right-handpart of the genome, that contains the cap gene encoding the capsidproteins of the virus.

AAV vectors may be prepared using standard methods in the art.Adeno-associated viruses of any serotype are suitable (see, e.g.,Blacklow, pp. 165-174 of “Parvoviruses and Human Disease” J. R.Pattison, ed. (1988); Rose, Comprehensive Virology 3:1, 1974; P.Tattersall “The Evolution of Parvovirus Taxonomy” In Parvoviruses (J RKerr, S F Cotmore. M E Bloom, R M Linden, C R Parrish, Eds.) p5-14,Hudder Arnold, London, U K (2006); and D E Bowles, J E Rabinowitz, R JSamulski “The Genus Dependovirus” (J R Kerr, S F Cotmore. M E Bloom, R MLinden, C R Parrish, Eds.) p15-23, Hudder Arnold, London, UK (2006), thedisclosures of each of which are hereby incorporated by reference hereinin their entireties). Methods for purifying for vectors may be found in,for example, U.S. Pat. Nos. 6,566,118, 6,989,264, and 6,995,006 andWO/1999/011764 titled “Methods for Generating High Titer Helper-freePreparation of Recombinant AAV Vectors”, the disclosures of which areherein incorporated by reference in their entirety. Methods of preparingAAV vectors in a baculovirus system are described in, e.g., WO2008/024998. AAV vectors can be self-complementary or single-stranded.Preparation of hybrid vectors is described in, for example, PCTApplication No. PCT/US2005/027091, the disclosure of which is hereinincorporated by reference in its entirety. The use of vectors derivedfrom the AAVs for transferring genes in vitro and in vivo has beendescribed (See e.g., International Patent Application Publication Nos.:91/18088 and WO 93/09239; U.S. Pat. Nos. 4,797,368, 6,596,535, and5,139,941; and European Patent No.: 0488528, all of which are herebyincorporated by reference herein in their entireties). Thesepublications describe various AAV-derived constructs in which the repand/or cap genes are deleted and replaced by a gene of interest, and theuse of these constructs for transferring the gene of interest in vitro(into cultured cells) or in vivo (directly into an organism). Thereplication defective recombinant AAVs according to the presentdisclosure can be prepared by co-transfecting a plasmid containing thenucleic acid sequence of interest flanked by two AAV inverted terminalrepeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes(rep and cap genes), into a cell line that is infected with a humanhelper virus (for example an adenovirus). The AAV recombinants that areproduced are then purified by standard techniques.

In some embodiments, the vector(s) for use in the methods of the presentdisclosure are encapsidated into a virus particle (e.g. AAV virusparticle including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, andAAV16). Accordingly, the present disclosure includes a recombinant virusparticle (recombinant because it contains a recombinant polynucleotide)comprising any of the vectors described herein. Methods of producingsuch particles are known in the art and are described in U.S. Pat. No.6,596,535, the disclosure of which is hereby incorporated by referencein its entirety.

Various exemplary embodiments of the invention are described herein.Reference is made to these examples in a non-limiting sense. They areprovided to illustrate more broadly applicable aspects of the invention.Various changes may be made to the invention described and equivalentsmay be substituted without departing from the true spirit and scope ofthe invention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processact(s) or step(s) to the objective(s), spirit or scope of the presentinvention. Further, as will be appreciated by those with skill in theart that each of the individual variations described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinventions. All such modifications are intended to be within the scopeof claims associated with this disclosure.

Any of the devices described for carrying out the subject diagnostic orinterventional procedures may be provided in packaged combination foruse in executing such interventions. These supply “kits” may furtherinclude instructions for use and be packaged in sterile trays orcontainers as commonly employed for such purposes.

The invention includes methods that may be performed using the subjectdevices. The methods may comprise the act of providing such a suitabledevice. Such provision may be performed by the end user. In other words,the “providing” act merely requires the end user obtain, access,approach, position, set-up, activate, power-up or otherwise act toprovide the requisite device in the subject method. Methods recitedherein may be carried out in any order of the recited events which islogically possible, as well as in the recited order of events.

Exemplary aspects of the invention, together with details regardingmaterial selection and manufacture have been set forth above. As forother details of the present invention, these may be appreciated inconnection with the above-referenced patents and publications as well asgenerally known or appreciated by those with skill in the art. Forexample, one with skill in the art will appreciate that one or morelubricious coatings (e.g., hydrophilic polymers such aspolyvinylpyrrolidone-based compositions, fluoropolymers such astetrafluoroethylene, hydrophilic gel or silicones) may be used inconnection with various portions of the devices, such as relativelylarge interfacial surfaces of movably coupled parts, if desired, forexample, to facilitate low friction manipulation or advancement of suchobjects relative to other portions of the instrumentation or nearbytissue structures. The same may hold true with respect to method-basedaspects of the invention in terms of additional acts as commonly orlogically employed.

In addition, though the invention has been described in reference toseveral examples optionally incorporating various features, theinvention is not to be limited to that which is described or indicatedas contemplated with respect to each variation of the invention. Variouschanges may be made to the invention described and equivalents (whetherrecited herein or not included for the sake of some brevity) may besubstituted without departing from the true spirit and scope of theinvention. In addition, where a range of values is provided, it isunderstood that every intervening value, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention.

Also, it is contemplated that any optional feature of the inventivevariations described may be set forth and claimed independently, or incombination with any one or more of the features described herein.Reference to a singular item, includes the possibility that there areplural of the same items present. More specifically, as used herein andin claims associated hereto, the singular forms “a,” “an,” “said,” and“the” include plural referents unless the specifically stated otherwise.In other words, use of the articles allow for “at least one” of thesubject item in the description above as well as claims associated withthis disclosure. It is further noted that such claims may be drafted toexclude any optional element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely,” “only” and the like in connection with the recitation of claimelements, or use of a “negative” limitation.

Without the use of such exclusive terminology, the term “comprising” inclaims associated with this disclosure shall allow for the inclusion ofany additional element—irrespective of whether a given number ofelements are enumerated in such claims, or the addition of a featurecould be regarded as transforming the nature of an element set forth insuch claims. Except as specifically defined herein, all technical andscientific terms used herein are to be given as broad a commonlyunderstood meaning as possible while maintaining claim validity.

The breadth of the present invention is not to be limited to theexamples provided and/or the subject specification, but rather only bythe scope of claim language associated with this disclosure.

1. A method for stimulating a tissue structure comprising lightsensitive protein, comprising: a. selecting a targeted tissue structureto be stimulated using illumination at a first subcutaneous location; b.providing an implantable light conductor configured to be permanentlycoupled between the first subcutaneous location and a second locationselected such that extracorporeal photons directed toward the secondlocation will be transmitted, at least in part, through the implantablelight conductor to the targeted tissue structure.